Carbazole EHop-016 derivatives as anticancer and anti-migratory agents

ABSTRACT

A series of novel EHop-016 derivatives is presented herein via designing and synthesizing compounds that mimics its more favorable “U-shaped” conformation that appears to be critical for inhibitory activity against Rac. Based on modeling studies on EHop-016, compounds with a more rigid structural conformation can mimic this “U-shaped” conformation and would improve the anti-migration activity against metastatic cells. Compounds are disclosed that inhibit RhoGTPases that are useful for inhibiting hyperprofilerative and neoplastic diseases, for instance compounds of formula 
     
       
         
         
             
             
         
       
     
     Specifically, the compounds inhibit the GTPases Rac and Cdc42 that are overactive or overexpressed in signaling pathways in cancer and metastasis. Methods for treatment of cancer and hyperproliferative diseases are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/168,612, filed Oct. 23, 2018, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.62/577,305, filed Oct. 26, 2017. The disclosures set forth in thereference applications are incorporated herein by reference in theirentireties.

This invention was made with Government support under Award Nos.NIH-NIGMS P20GM103475-13 and NIH-NIGMS SC3GM116713 awarded by TheNational Institutes of Health. The U.S. Government has certain rights inthe invention.

BACKGROUND

Many compounds are disclosed that inhibit Rho GTPases that are usefulfor inhibiting hyperprofilerative and neoplastic diseases. Specifically,the compounds inhibit the GTPases Rac and Cdc42 that are overactive oroverexpressed in signaling pathways in cancer and metastasis. Methodsfor treatment of cancer and hyperproliferative diseases are disclosed.

The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42(cell division control protein 42 homolog) regulate cell functionsgoverning cancer malignancy, including cell polarity, migration, andcell cycle progression. The Rho family of GTPases in humans consists of20 different members, and aberrant behavior in their regulatory activityhas been implicated in cancer and other diseases. More than 70 Guaninenucleotide Exchange Factors (GEFs) are known, which specificallyactivate one or more of the GTPases. In turn, the activated GTPases canspecifically interact with over 60 downstream effectors. Dysregulationof one or more cellular processes can lead to release of malignant cellsfrom their original locations, which subsequently can establishthemselves in pre-metastatic niches in, for example, bone or lungs. Ithas been found that members of the Rho GTPase family, including Rac,Cdc42 and Rho, play key signaling roles in these processes.

Rho GTPases regulate migration and invasion, cytoskeletal organization,transcriptional regulation, cell cycle progression, apoptosis, vesicletrafficking, and cell-to-cell and cell-to-extracellular matrixadhesions. The Rho GTPases Rac and Cdc42 are potent inducers of actinpolymerization and extension of actin structures at the leading edge ofmotile cells. In addition, Cdc42 plays a critical role in cell polarity,and thus, promotes directed and persistent migration.

Studies have implicated hyperactive Rac and Cdc42 with increased cancercell survival, proliferation, and invasion, as well in Ras and otheroncogene-mediated transformation. Furthermore, oncogenic cell surfacereceptors, such as tyrosine kinase, cytokine, and G protein coupledreceptors, activate Rac and Cdc42 via regulation of their upstreameffector GEFs. Accordingly, Rae and Cdc42 proteins are generally notmutated in cancer but rather overexpressed or hyperactivated. Eventhough ˜9% of melanomas contain an activating Rac(P29S) mutation, andthe hyperactive splice variant Rac1b is overexpressed in some cancers, amajority of the Rac and Cdc42 in human cancer are activated due toupregulated GEFs.

Of the direct downstream effectors of Rae and Cdc42, p21-activatedkinases (PAK) are overexpressed in a number of cancers and contribute tocancer transformation and progression by regulating key cellularfunctions, including cytoskeletal organization, cell migration,adhesion, growth, and development. Therefore, a number of PAK inhibitorshave been developed as anti-cancer therapeutics. However, these havebeen limited by specificity, bioavailability, and toxicity, and have yetto successfully complete clinical trials.

In cancer patients, the ability of tumor cells to spread from primarytumors (and metastatic tumors) is the major cause of death. In the casethat breast cancer is detected and treated prior to metastasis, thepatient has higher probability of being cured of their disease. Tosuccessfully invade a secondary site, a cancer cell must complete aseries of steps including separation from primary tumor (intravasation),invasion through surrounding tissues and basement membranes, entry andsurvival in the circulation, and arrest in a distant target organ(extravasation). During cancer cell invasion, tumor cell migrationthrough tissues, frequently requires the degradation of theextracellular matrix (ECM) and several proteins play a key role in thisprocess. Moreover, invadopodia are actin-rich protrusive structures withassociated matrix degradation activity and are believed to be importantfor tumor cells to penetrate the basement membrane of epithelia andblood vessels. The small GTPase Rac1, a member of the Ras superfamily ofGTPases has been implicated in regulating migration and invasion ofbreast cancer cells. Rac1 is activated by GTP/GDP exchange factors (GEF)that are regulated via a myriad of cell surface receptors. Therefore,therapeutic strategies targeting the binding of Rac1 to GEFs are arational means to inhibit migration of cancer cells.

The carbazole skeleton contained in many synthetic and natural compoundsis the key structural motif of many biological activities that arerelated to this type of molecules.

Many carbazole derivatives are well known for their variouspharmacological activities such as antioxidant, anti-inflammatories,antibacterial, antitumor, anticonvulsant, antipsychotics, antidiabetics,and larvicidal properties. The cytotoxic activity of carbazole alkaloidshas been related to their polycyclic, planar and aromatic structure.Some carbazole derivatives have been evaluated for their anti-tumorpotential against several human tumor cell lines. The carbazolesulfonamide IG-105 is an antimitotic agent that inhibit microtubuleassembly through specific interactions within tubulin structure.Modeling studies suggest that the dimethoxypyridine moiety and thecarbazole group forms interactions with the hydrophobic pocket oftubulin, and that the sulfonamino group and the N atom of the carbazoleforms hydrogen bonds. The HYL-6d derivative inhibits angiogenicproliferation and migration in HUVEC cells under pathological angiogenicconditions, which is critical in breast cancer progression andmetastasis formation. The MHY407 is an epoxypropoxy carbazole derivativethat effectively causes DNA damage by C-PARP production, topoisomeraseII inhibition and cell cycle arrest at S phase by regulating cyclin Dl,pRb, and p21 levels.

EHop-016 has recently been developed and inhibits metastatic cancer cellviability at concentrations of >5 μM. Results showed that EHop-016inhibits Rac1-Vav2 interaction with IC50=1 μM, Rac1 downstream effectorPAK1 by −60% at 2 μM, lamellipodia formation, and cell migration.EHop-016 and other compounds are described in PCT International PatentApplication No. PCT/US2017/029921 (filed Apr. 27, 2017), which is hereinincorporated by reference in its entirety.

There is a need for new therapeutic agents for the treatment of cancerand other hyperproliferative diseases. The Rac and Cdc42 GTPases areimportant cellular mediators that are hyperactive or overexpressed inmetastatic tumors. Design of novel inhibitors of the activities of Racand/or Cdc42 with improved activity, pharmacochemical profile andreduced toxicity is desirable.

SUMMARY

A summary of the results of characteristics of novel EHop-016derivatives HV107 and HV118 is:

-   -   1. HV-107 and HV-118 are toxic to metastatic breast cancer cells        MDA-MB-231 and MDA-MB-435 (but not non-cancerous cells);    -   2. HV-107 and HV-118 inhibit RAC;    -   3. HV-107 and HV-118 inhibit cell survival by promoting        apoptosis (caspase 3 induction);    -   4. HV-107 and HV-118 promote cell survival signaling (inhibit        Akt phosphorylation);    -   5. HV-107 and HV-118 inhibit cell migration;    -   6. HV-118 reduces tumor growth and metastasis to lung and liver        in mice; and    -   7. HV-118 is not toxic to mice

A series of novel of EHop-016 derivatives is presented herein viadesigning and synthesizing compounds that mimics its more favorable“U-shaped” conformation that appears to be critical for inhibitoryactivity against Rac. Based on modeling studies on EHop-016, compoundswith a more rigid structural conformation can mimic this “U-shaped”conformation would improve the anti-migration activity againstmetastatic cells.

The present disclosure provides other more potent inhibitors of Raccompared to EHop-016. For instance, the first two series of compounds(3a-d and 4a-f, scheme 1; see FIG. 1) the replacement of the corepyrimidine group in EHop-016 with a pyridine, was explored whichsubstitution at positions-2 and -3 with aliphatic amines and thecarbazole group, respectively (or vice versa), place the keypharmacophores from EHop-016 in a more rigid 1,2-substitution sequence.Compounds 3a-d were synthesized by initial amide formation reacting2-chloronicotinic acid 1 with carbazole 2, following by Cul-catalyzedcoupling reaction with various amines to afford the corresponding2-aminonicotinamide derivatives 3a-d. On the other hand, compounds 4a-fwere synthesized by reacting 2-chloronicotinic acid 1 and carbazole 2were under microwave irradiation at 140° C. for 5 h using 3 equiv. ofDIPEA in water (scheme 1). The product 2-aminocarbazolenicotinic acidintermediate was reacted with various amines to afford the corresponding2-aminocarbazole-nicotinamde derivatives. The growth inhibitory potencyagainst MCF-7 and MDA-Mb-231 breast cancer cells was tested.

The following numbered embodiments are contemplated and arenon-limiting:

1. A compound of formula (I),

wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl; wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl isindependently optionally substituted;

-   -   each R² is independently deuterium, halogen, —OH, —CN, C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₁-C₆ to        aryl, —OR⁵, —C(O)OR⁵, —C(O)NR⁵R⁶, —NH₂, —NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl,        —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆        alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆        alkyl), —S(O)₂NH(C₁-C₆ alkyl); wherein each hydrogen atom in        C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, and        C₆C₁₀ aryl is independently optionally substituted;    -   A is —O—, —S—, —N(R⁷)—, —N(R⁷)C(O)—, or —C₁-C₆ alkyl-, wherein        each hydrogen atom in C₁-C₆ alkyl is optionally substituted;    -   the dashed bond is either present so that the bond is a double        bond or absent so that the bond is a single bond;    -   X is ═C(R⁸)—, ═N—, —N═, or —C(R⁸)₂—, Y is ═C(R⁹)—, ═N—, —N═, or        —C(R⁹)₂—, and Z is ═C(R¹)—, —C(R¹⁰)═, ═N—, —N═, or —C(R¹⁰)₂—;        provided that when X is ═C(R⁸)— or ═N—, Z is not ═C(R¹⁰)— or        —═N—, when Y is ═C(R⁹)— or ═N—, Z is not ═C(R¹⁰)— or —═N—,    -   each R³ is independently deuterium, halo, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆        alkyl-(NHR¹¹), C₁-C₆ alkyl-(OR¹¹), C₆-C₁₀ aryl-(OR¹¹), C₁-C₆        alkyl-(NR¹¹R¹²), —NHR¹¹, —NR¹¹R¹², —OR¹¹, —C(O)C₁-C₆ alkyl,        —C(O)R¹¹, or mono- or bicyclic heteroaryl; wherein each hydrogen        atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl,        C₆-C₁₀ aryl-(C₆-C₁₀ aryl), C₁-C₆ alkyl-(NHR¹¹), C₁-C₆        alkyl-(OR¹¹), C₆-C₁₀ aryl-(OR¹¹), C₁-C₆ alkyl-, —C(O)C₁-C₆        alkyl, or mono- or bicyclic heteroaryl, is optionally        substituted by deuterium, halogen, —OH, oxo, —OR¹³, —NHR¹³, —CN,        —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl), —NH(C₆-C₁₀ aryl),        —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl, —N(C₁-C₆        alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl,        —NHC(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂,        —NHC(O)OC₁-C₆ alkyl, —N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl,        —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆ alkyl), —N(C₁-C₆        alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆ alkyl),        —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆        alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),        —NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆        alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl),        —N(C₁-C₆ alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆        alkyl)₂, —CO₂H, —C(O)C₁-C₆ alkyl, —C(O)OC₁-C₆ alkyl, —C(O)NH₂,        —C(O)NH(C₁-C₆ alkyl), —C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl,        —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆ alkyl, —S(O)NH(C₁-C₆ alkyl),        —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆ alkyl)₂, —S(O)₂N(C₁-C₆        alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂, C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3- to 7-membered        heterocycloalkyl), —CF₃, —CHF₂, or —CH₂F;    -   each R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² is independently H,        deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₁-C₆ heteroalkyl, 3- to 7-membered        heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3- to 7-membered        heterocycloalkyl), heteroaryl, —OC₁-C₆ alkyl, —NHC₁-C₆ alkyl,        —N(C₁-C₆ alkyl)₂, —S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or —CH₂F,        wherein each hydrogen in C₁-C₆alkyl, C₁-C₆heteroalkyl, and        C₆-C₁₀ aryl is optionally substituted by —O—C₁-C₆ alkyl, C₃-C₆        cycloalkyl, 3- to 7-membered heterocycloalkyl, 3- to 7-membered        substituted heterocycloalkyl, —CH₂CN, —N(C₁-C₆ alkyl)₂, —OC₁-C₆        alkyl, optionally substituted aryl, —C(O)NH-optionally        substituted aryl, hydroxy, or mono- or bicyclic heteroaryl; and    -   a is 0, 1, 2, or 3,    -   b is 0, 1, or 2,

or a salt thereof.

2. The compound or a salt thereof of clause 1, wherein the compound isof the formula

-   -   wherein each of R³, R^(3′), and R³″ are as defined for R³ above.

3. The compound or a salt thereof of clause 1 or clause 2, wherein thedashed bond is a double bond and X is ═C(R⁸)—, Y is ═N—, and Z is—C(R¹⁰)═; X is ═N—, Y is ═N—, and Z is —C(R¹⁰)═; X is ═N—, Y is ═C(R⁹)—,and Z is —N═; X is ═N—, Y is ═C(R⁹)—, and Z is —C(R¹⁰)═; or X is ═N—, Yis ═C(R⁹)—, and Z is —C(R¹⁰)═; or the dashed bond is absent and X is—C(Rx)₂-, Y is —C(R⁹)₂—, and Z is —C(R¹⁰)₂—;

wherein each R⁸, R⁹, and R¹⁰ are each individually H, deuterium, halo,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl; wherein each hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl,C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl is independently optionallysubstituted.

4. The compound or a salt thereof of clause 1, wherein the compound hasthe structure

5. The compound or salt thereof of clause 2, wherein the compound hasthe structure

6. A method of treating a disease in a patient, the method comprisingadministering to the patient in need thereof an effective amount of acompound according to any one of clauses 1 to 5.

7. The method of clause 6, wherein the disease is cancer.

8. The method of clause 7, wherein the compound inhibits cancer cellmigration.

9. The method of clause 7, wherein the cancer is selected from the groupconsisting of breast cancer, pancreatic cancer, ovarian cancer, gastriccancer, and neuronal cancer.

10. The method of clause 9, wherein the cancer is pancreatic cancer.

-   -   11. The method of clause 9, wherein the cancer is ovarian        cancer.    -   12. The method of clause 9, wherein the cancer is gastric        cancer.    -   13. The method of clause 9, wherein the cancer is neuronal        cancer.    -   14. The method of clause 9, wherein the cancer is breast cancer.    -   15. The method of clause 14, wherein the compound inhibits        mammosphere formation.    -   16. The method of any one of clauses 6 to 15, wherein the        compound induces cell cycle arrest of a diseased cell.    -   17. The method of any one of clauses 6 to 16, wherein the        compound induces apoptosis of a diseased cell.    -   18. The method of any one of clauses 6 to 17, wherein the        compound reduces the expression of a Bcl-2 protein.    -   19. The method of any one of clauses 6 to 18, wherein the        disease is mediated by a GTPase.    -   20. The method of clause 19, wherein the GTPase is Rac 1 or        Cdc42.    -   21. The method of clause 20, wherein the GTPase is Rac1.    -   22. The method of clause 20, wherein the GTPase is Cdc42.    -   23. The method of any one of clauses 6 to 22, wherein the        compound inhibits PAK1/2 activity.    -   24. The method of any one of clauses 6 to 23, wherein the        compound inhibits STAT3 activity.    -   25. The method of any one of clauses 6 to 24, wherein the        effective amount of the compound is in a range of about 0.01        mg/kg to about 100 mg/kg of body weight of the patient.    -   26. The method of any one of clauses 6 to 24, wherein the        effective amount of the compound is in a range of about 0.1        mg/kg to about 50 mg/kg of body weight of the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Design and synthesis “Scheme 1”. General synthetic routes of2-substituted-nicotinamide derivatives 3a-d and 4a-f. Reagents andconditions: (a) (i) HOBt, ED AC, DMF, EtsN, rt; (h) Cul, DIPEA, dioxane,80° C., 8-10 h, R═HNR¹R²; (b) Method A (1) DIPEA (3 equiv), water, 140°C., 5 h, (n) HOBt, ED AC, DMF, EtsN, rt, R—NH₂. Method B (1) HOBt, EDAC, CH₂C₁₂, EtsN, rt; (11) Cul, CS₂CO₃, DMSO, 90° C., 24 h, R—NH₂.

FIG. 2. Design and synthesis “Scheme 2”. General synthetic routes to2,3-diamino-substituted pyridines 7a-c and 11a-b. Reagents andconditions: (a) THF, Et3N, reflux, 2 h; (b) (i) SnCk, HCl, EtOH, reflux,(ii) HOBt, EDAC, DMF, EtsN, rt (for 7a) or THF, EtsN, reflux (for 7b-c);(c) R═HNR¹R², THF, EtsN, reflux; (d) SnCk, HCl, EtOH, reflux; (e) 10,HOBt, EDAC, DMF, EtsN, rt (for 11a-b).

FIG. 3. Design and synthesis “Scheme 3”. General synthetic route toortho-diamide derivatives 14a-h. Reagents and conditions: (a) THF, rt 16h; (b) HOBt, EDC, THF, rt, 16 h.

FIG. 4 (A-B). Log-dose response curve for compounds 4a, 4c, 4d, 4e (FIG.4A), 7a, 7c, and 11a (FIG. 4B) in MCF-7 cancer cells. Each data pointrepresents the mean of three (3) replicates, error bars represent 95%confidence intervals. Each GI₅₀ was calculated based on sigmoidal curvefitting to the respective data set.

FIG. 5 (A-B). Log-dose response curve for compounds 3d, 4d, 4e (FIG.5A), 7a, 7b, and 11a (FIG. 5B) in MDA-MB-231 cancer cells. Each datapoint represents the mean of three (3) replicates, error bars represent95% confidence intervals. Each GI₅₀ was calculated based on sigmoidalcurve fitting to the respective data set.

FIG. 6. Wound Healing Assay. Inhibitory effect of 11b and 3b onMDA-MB-231 cells migration detected by wound-healing assay. MDA-MB-231cells were treated with vehicle or with 11b and 3b at 10 pM andphotomicrograph obtained at 0, 12, and 24 h.

FIG. 7. Effect of HV-107 on cell viability. Cells were treated withvehicle control (0.5% DMSO) or varying concentrations of HV-107(250-6000 nM) for 48 h. Cell viability was measured using the CellTiter96® AQueous One Solution Cell Proliferation Assay from Promega, as permanufacturer's instruction. Relative cell viability is presented fornon-cancerous mammary epithelial cells (MCF10A) or metastatic breastcancer cells MDA-MB-231 or MDA-MB-435. N=3-4, each with three technicalreplicates; Error bars represent ±SEM; * p≤0.05.

FIG. 8 (A-B). Effect of HV-107 on Rac activation. FIG. 8A. MDA-MB-231and FIG. 8B. MDA-MB-435 human breast cancer cells were treated for 24 hwith 0, 100, or 250 nM HV-107. After treatment, total protein wasextracted and equal amounts of proteins subjected to pulldown assaysusing the p21-binding domain of PAK to isolate the GTP bound (active)Rac. Samples were then western blotted for total and active Rac andpositive bands quantified using image J. N=2; error bars represent±SEM; * p≤0.05.

FIG. 9. Effect of HV-118 on cell viability. Cells were treated withvehicle control (0.5% DMSO) or varying concentrations of HV-118 (10-500nM) for 48 h. Cell viability was measured using the CellTiter 96®AQueous One Solution Cell Proliferation Assay from Promega, as permanufacturer's instruction. Relative cell viability is presented forhuman non-cancerous mammary epithelial cells (MCF10A) or humanmetastatic breast cancer cells MDA-MB-231 or MDA-MB-435. N=3, each withthree technical replicates; error bars represent ±SEM; * p≤0.05.

FIG. 10 (A-B). Effect of HV-118 on Rac activation. FIG. 10A. MDA-MB-231and FIG. 10B. MDA-MB-435 human breast cancer cells were treated for 24 hwith 0, 25, 50, or 100 nM HV-118. After treatment, total protein wasextracted and equal amounts of proteins subjected to pulldown assaysusing the p21-binding domain of PAK to isolate the GTP bound (active)Rac. Samples were then western blotted for total and active Rac andpositive bands quantified using image J. N=3-6; error bars represent±SEM; * p≤0.05.

FIG. 11 (A-B). Synthesis of EHop-016 derivatives with modifications inbuilding block C. FIG. 11A shows Scheme 1B, a two-step syntheticprocedure for the synthesis of EHop-016 and derivatives. FIG. 11B showsScheme 2B, a procedure for the synthesis of additional EHop-016derivatives.

FIG. 12 (A-B). Migration Assays for Compounds 5, 10, and 11 compared toEHop-016. Migration assays were performed via the Scratch Method. FIG.12A shows photographs obtained with EHop-016 at concentrations of 1.0and 5.0 μM. FIG. 12B shows a comparison of migration assays forCompounds 5, 10, and 11.

FIG. 13. Scheme 3B. Synthesis of HV-118 and EHop-016 derivative 8.

FIG. 14 (A-B). Log dose-response curve for compound 8 in MCF-7 (FIG.14A) and SH-SY5Y neuroblastoma (FIG. 14B) cells.

FIG. 15 (A-D). Effect of HV-107 and HV-118 on Caspase 3/7 activity. FIG.15 A, C. MDA-MB-231 and FIG. 15 B, D. MDA-MB-435 human metastatic breastcancer cells were treated for 48 or 72 h with 0, 500 or 1000 nM HV-107,100 or 200 nM HV-118. Caspase 3/7 activity was measured using theCaspase-Glo 3/7 Assay (Promega, Corp.) as per manufacturer'sinstruction. N=4: error bars represent ±SEM; * p≤0.05.

FIG. 16 (A-D). Inhibition of pAKT by HV-107 and HV-118. FIG. 16 A, C.MDA-MB-231 and FIG. 16 B, D. MDA-MB-435 human metastatic breast cancercells were treated for 24 h with 0, 250, 500 or 1000 nM HV-107, 100 or200 nM HV-118. After treatment, total protein was extracted, and equalamounts of proteins were western blotted for total and phosphorylatedAkt (pAkt). Positive bands were quantified using image J. N=4-6; errorbars represent t SEM; * p≤0.05.

FIG. 17 (A-B). Effect of HV-107 and HV-118 on cell migration. Woundswere created on confluent monolayers of MDA-MB-231 or MDA-MB-435 humanmetastatic breast cancer cells. Immediately after, cells were treatedfor 24 h with FIG. 17A. 0, 1000, or 2000 nM of HV-107; and FIG. 17B. 0,100, or 200 nM HV-118. The distance traveled by HV-107 and HV-118treated cells that migrated to close the wound was quantified relativeto the distance traveled by control-treated cells. N=4-7; error barsrepresent ±SEM; * p≤0.05.

FIG. 18 (A-D). Effect of HV-118 on mammary tumor growth. Severe combinedimmune deficiency (SCID) mice were inoculated at the mammary fat padwith GFP-MDA-MB-231 cells and treated with 0 (Vehicle) or 1 mg/kg BWHV-118 (5× a wk). FIG. 18A. Upper panel, representative images and lowerpanel, average relative tumor growth on last day. FIG. 18B. Relativetumor growth as a function of days following HV-118 administration.Lungs and livers were removed at necropsy and imaged for fluorescentmetastatic foci. FIG. 18C. Representative organs under fluorescencemicroscopy for 0 (Vehicle) and 1 mg/kg BW HV-118 treatment. FIG. 18D.Relative average integrated intensity of fluorescent metastaticfoci/organ/treatment. N=13-15 mice/group; error bars represent ±SEM.

FIG. 19 (A-C). HV-118 toxicity assessment. Blood collected at necropsywas used for FIG. 19A. complete blood count (CBC) analysis and FIG. 19B.determination of serum levels of liver enzymes Alanine Aminotranferase(ALT), Aspartate Aminotransferse (AST), and Alkaline phosphatase (ALP).N=4-5 mice/group; error bars represent ±SEM. FIG. 19C. Average mouseweight as a function of days following HV-118 administration. N=13-15mice/group.

DETAILED DESCRIPTION

The present disclosure provides novel Ehop-016 derivatives and assessestheir efficacy to inhibit Rac activation and selectively inhibitmetastatic breast cancer cell viability.

Novel Rac Inhibitors as Targeted Therapy for Metastatic Breast Cancer

Effective therapeutics for metastatic breast cancer are disclosed bytargeting the Rho GTPase Rac. Metastatic disease is the primary cause ofbreast cancer mortality, but effective treatments remain elusive.Therefore, there is a critical need to develop efficient strategies toinhibit metastatic breast cancer. The Rho GTPase Rac is a key molecularswitch activated by a myriad of cell surface receptors to promote cancercell migration/invasion, proliferation, and survival. Metastatic breastcancers often overexpress or exhibit high Rac activity. Therefore, therationale for the proposed research is that inhibition of theinteraction of Rac with their upstream effectors, guanine nucleotideexchange factors (GEFs), will impede cancer progression.

EHop-016 is a small molecule that inhibits Rac activity of metastaticbreast cancer cells with an IC50 of 1 μM. EHop-016 is 10-100 times moreactive than previously available Rac inhibitors, and is the firstcompound shown to inhibit the activation of Rac by the oncogenic GEFVav. EHop-016 inhibits the activity of the Rac downstream effector p21activated kinase (PAK), extension of actin structures, and cellmigration of metastatic breast cancer cells. At higher concentrations(≥10 μM) EHop-016 also inhibits cell viability and the related proteinCdc42 activity. EHop-016 at ≥25 mg/kg Body Weight (BW) significantlyreduced tumor growth, metastasis, and angiogenesis in a mouse model.Moreover, the utility of EHop-016 as a Rac inhibitor has been shown invitro and in vivo in a range of cancer types such as leukemia, prostatecancer, and melanoma. In addition to affecting cancer cells, EHop-016may also inhibit the tumor promoting immune cells in the tumormicroenvironment. However, pharmacokinetic study of EHop-016 in a mousemodel demonstrated that EHop-016 was cleared rapidly from thecirculation and that the bioavailability of this inhibitor needs to beimproved for further pharmacological development. A panel of Ehop-016derivatives was developed and tested. HV-107 inhibits Rac activation by50% at 250 nM in breast cancer cells. Moreover, HV-107 inhibits breastcancer cell viability by 45% and is not toxic to non-cancerous mammaryepithelial cells at concentrations ≥1 μM.

To demonstrate that HV-107 will inhibit metastatic breast cancerprogression more efficiently than Ehop-016, the molecular mechanisms ofHV-107 in breast cancer cells was examined.

Elucidation of the Molecular Mechanisms of HV-107.

The experimental approach to identify the mechanism of action of HV-107is to: 1) characterize the effects of HV-107 on Rac-regulated cellfunctions; and 2) determine the activation status of known downstreamtargets of Rac using MDA-MB-231 and MDA-MB-435 metastatic breast cancercells.

HV-107 is as a novel chemical probe with increased potency against Rac.

Characterization of the Rac Inhibitor HV-118 as Targeted Therapy forMetastatic Breast Cancer

A panel of Ehop-016 derivatives was tested. HV-118 inhibits Racactivation by 65% at 100 nM in breast cancer cells. Moreover, HV-118inhibits breast cancer cell viability, while showing minimal toxicitytowards non-cancerous mammary epithelial cells. Therefore, preliminarydata suggest HV-118 could be up to 20 times more potent than EHop-016.

To demonstrate that HV-118 will inhibit metastatic breast cancerprogression more efficiently than Ehop-016, the molecular mechanisms ofHV-118 in breast cancer cells was examined, and its efficacy to inhibitcancer progression validated in a mouse model of metastatic breastcancer.

Elucidation of the Molecular Mechanisms of HV-118.

To identify the mechanism of action of HV-118: 1) the effects of HV-118on Rac-regulated cell functions were characterized; and 2) theactivation status of known downstream targets of Rac using MDA-MB-231and MDA-MB-435 metastatic breast cancer cells was determined.

Validation of HV-118 as an Inhibitor of Metastatic Breast CancerProgression In Vivo.

Preliminary data supporting HV-118 as a potent inhibitor of Racactivation in breast cancer cells was confirmed. The in vivo efficacy ofHV-118 is identified by testing HV-118 in immunocompromised miceinoculated with green fluorescent protein (GFP)-tagged MDA-MB-231 cells.Tumor growth and metastasis were assessed by fluorescent imaging. HV-118toxicity was also determined.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense.

To provide a more concise description, some of the quantitativeexpressions given herein are not qualified with the term “about”. It isunderstood that, whether the term “about” is used explicitly or not,every quantity given herein is meant to refer to the actual given value,and it is also meant to refer to the approximation to such given valuethat would reasonably be inferred based on the ordinary skill in theart, including equivalents and approximations due to the experimentaland/or measurement conditions for such given value. Whenever a yield isgiven as a percentage, such yield refers to a mass of the entity forwhich the yield is given with respect to the maximum amount of the sameentity that could be obtained under the particular stoichiometricconditions. Concentrations that are given as percentages refer to massratios, unless indicated differently.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

Chemical nomenclature for compounds described herein has generally beenderived using the commercially-available ACD/Name 2014 (ACD/Labs) orChemBioDraw Ultra 13.0 (Perkin Elmer).

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentdisclosure and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

As used herein, the term “alkyl” includes a chain of carbon atoms, whichis optionally branched and contains from 1 to 20 carbon atoms. It is tobe further understood that in certain embodiments, alkyl may beadvantageously of limited length, including C₁-C₁₂, C₁-C₁₀, C₁-C₉,C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄, Illustratively, such particularlylimited length alkyl groups, including C₁-C₈, C₁-C₇, C₁-C₆, and C₁-C₄,and the like may be referred to as “lower alkyl.” Illustrative alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 2-pentyl,3-pentyl, neopentyl, hexyl, heptyl, octyl, and the like. Alkyl may besubstituted or unsubstituted. Typical substituent groups includecycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy,mercapto, alkylthio, arylthio, cyano, halo, carbonyl, oxo, (═O),thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, C-carboxy, O-carboxy, nitro, and amino, or asdescribed in the various embodiments provided herein. It will beunderstood that “alkyl” may be combined with other groups, such as thoseprovided above, to form a functionalized alkyl. By way of example, thecombination of an “alkyl” group, as described herein, with a “carboxy”group may be referred to as a “carboxyalkyl” group. Other non-limitingexamples include hydroxyalkyl, aminoalkyl, and the like.

As used herein, the term “alkenyl” includes a chain of carbon atoms,which is optionally branched, and contains from 2 to 20 carbon atoms,and also includes at least one carbon-carbon double bond (i.e. C═C). Itwill be understood that in certain embodiments, alkenyl may beadvantageously of limited length, including C₂-C₁₂, C₂-C₉, C₂-C₈, C₂-C₇,C₂-C₆, and C₂-C₄. Illustratively, such particularly limited lengthalkenyl groups, including C₂-C₅, C₂-C₇, C₂-C₆, and C₂-C₄ may be referredto as lower alkenyl. Alkenyl may be unsubstituted, or substituted asdescribed for alkyl or as described in the various embodiments providedherein. Illustrative alkenyl groups include, but are not limited to,ethenyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like.

As used herein, the term “alkynyl” includes a chain of carbon atoms,which is optionally branched, and contains from 2 to 20 carbon atoms,and also includes at least one carbon-carbon triple bond (i.e. C≡C). Itwill be understood that in certain embodiments, alkynyl may each beadvantageously of limited length, including C₂-C₁₂, C₂-C₉, C₂-C₈, C₂-C₇,C₂-C₆, and C₂-C₄. Illustratively, such particularly limited lengthalkynyl groups, including C₂-C₈, C₂-C₇, C₂-C₆, and C₂-C₄ may be referredto as lower alkynyl. Alkenyl may be unsubstituted, or substituted asdescribed for alkyl or as described in the various embodiments providedherein. Illustrative alkenyl groups include, but are not limited to,ethynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl, and the like.

As used herein, the term “aryl” refers to an all-carbon monocyclic orfused-ring polycyclic groups of 6 to 12 carbon atoms having a completelyconjugated pi-electron system. It will be understood that in certainembodiments, aryl may be advantageously of limited size such as C₆-C₁₀aryl. Illustrative aryl groups include, but are not limited to, phenyl,naphthylenyl and anthracenyl. The aryl group may be unsubstituted, orsubstituted as described for alkyl or as described in the variousembodiments provided herein.

As used herein, the term “cycloalkyl” refers to a 3 to 15 memberall-carbon monocyclic ring, including an all-carbon 5-member/6-member or6-member/6-member fused bicyclic ring, or a multicyclic fused ring (a“fused” ring system means that each ring in the system shares anadjacent pair of carbon atoms with each other ring in the system) group,where one or more of the rings may contain one or more double bonds butthe cycloalkyl does not contain a completely conjugated pi-electronsystem. It will be understood that in certain embodiments, cycloalkylmay be advantageously of limited size such as C₃-C₁₃, C₃-C₉, C₃-C₆ andC₄-C₆. Cycloalkyl may be unsubstituted, or substituted as described foralkyl or as described in the various embodiments provided herein.Illustrative cycloalkyl groups include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclopentadienyl,cyclohexyl, cyclohexenyl, cycloheptyl, adamantyl, norbornyl,norbornenyl, 9H-fluoren-9-yl, and the like. Illustrative examples ofcycloalkyl groups shown in graphical representations include thefollowing entities, in the form of properly bonded moieties:

As used herein, the term “heterocycloalkyl” refers to a monocyclic orfused ring group having in the ring(s) from 3 to 12 ring atoms, in whichat least one ring atom is a heteroatom, such as nitrogen, oxygen orsulfur, the remaining ring atoms being carbon atoms. Heterocycloalkylmay optionally contain 1, 2, 3 or 4 heteroatoms. Heterocycloalkyl mayalso have one of more double bonds, including double bonds to nitrogen(e.g. C═N or N═N) but does not contain a completely conjugatedpi-electron system. It will be understood that in certain embodiments,heterocycloalkyl may be advantageously of limited size such as 3- to7-membered heterocycloalkyl, 5- to 7-membered heterocycloalkyl, and thelike. Heterocycloalkyl may be unsubstituted, or substituted as describedfor alkyl or as described in the various embodiments provided herein.Illustrative heterocycloalkyl groups include, but are not limited to,oxiranyl, thianaryl, azetidinyl, oxetanyl, tetrahydrofuranyl,pyrrolidinyl, tetrahydropyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl,1,4-dithianyl, piperazinyl, oxepanyl, 3,4-dihydro-2H-pyranyl,5,6-dihydro-2H-pyranyl, 2H-pyranyl, 1, 2, 3, 4-tetrahydropyridinyl, andthe like. Illustrative examples of heterocycloalkyl groups shown ingraphical representations include the following entities, in the form ofproperly bonded moieties:

As used herein, the term “heteroaryl” refers to a monocyclic or fusedring group of 5 to 12 ring atoms containing one, two, three or four ringheteroatoms selected from nitrogen, oxygen and sulfur, the remainingring atoms being carbon atoms, and also having a completely conjugatedpi-electron system. It will be understood that in certain embodiments,heteroaryl may be advantageously of limited size such as 3- to7-membered heteroaryl, 5- to 7-membered heteroaryl, and the like.Heteroaryl may be unsubstituted, or substituted as described for alkylor as described in the various embodiments provided herein. Illustrativeheteroaryl groups include, but are not limited to, pyrrolyl, furanyl,thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl,pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, tetrazolyl, triazinyl,pyrazinyl, tetrazinyl, quinazolinyl, quinoxalinyl, thienyl, isoxazolyl,isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,benzoxazolyl, benzthiazolyl, benzisoxazolyl, benzisothiazolyl andcarbazoloyl, and the like. Illustrative examples of heteroaryl groupsshown in graphical representations, include the following entities, inthe form of properly bonded moieties:

As used herein, “hydroxy” or ““hydroxyl” refers to an —OH group.

As used herein, “alkoxy” refers to both an —O-(alkyl) or an—O-(unsubstituted cycloalkyl) group. Representative examples include,but are not limited to, methoxy, ethoxy, propoxy, butoxy,cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and thelike.

As used herein, “aryloxy” refers to an —O-aryl or an —O-heteroarylgroup. Representative examples include, but are not limited to, phenoxy,pyridinyloxy, furanyloxy, thienyloxy, pyrimidinyloxy, pyrazinyloxy, andthe like, and the like.

As used herein, “mercapto” refers to an —SH group.

As used herein, “alkylthio” refers to an —S-(alkyl) or an—S-(unsubstituted cycloalkyl) group.

Representative examples include, but are not limited to, methylthio,ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio,cyclopentylthio, cyclohexylthio, and the like.

As used herein, “arylthio” refers to an —S-aryl or an —S-heteroarylgroup. Representative examples include, but are not limited to,phenylthio, pyridinylthio, furanylthio, thienylthio, pyrimidinylthio,and the like.

As used herein, “halo” or “halogen” refers to fluorine, chlorine,bromine or iodine.

As used herein, “cyano” refers to a —CN group.

The term “oxo” represents a carbonyl oxygen. For example, a cyclopentylsubstituted with oxo is cyclopentanone.

As used herein, “bond” refers to a covalent bond.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents. Where the term “substituted” isused to describe a structural system, the substitution is meant to occurat any valency-allowed position on the system. In some embodiments,“substituted” means that the specified group or moiety bears one, two,or three substituents. In other embodiments, “substituted” means thatthe specified group or moiety bears one or two substituents. In stillother embodiments, “substituted” means the specified group or moietybears one substituent.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance may but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “wherein each hydrogenatom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclicheteroaryl is independently optionally substituted by C₁-C₆ alkyl” meansthat an alkyl may be but need not be present on any of the C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl byreplacement of a hydrogen atom for each alkyl group, and the descriptionincludes situations where the C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, ormono- or bicyclic heteroaryl is substituted with an alkyl group andsituations where the C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3-to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl is not substituted with the alkyl group.

As used herein, “independently” means that the subsequently describedevent or circumstance is to be read on its own relative to other similarevents or circumstances. For example, in a circumstance where severalequivalent hydrogen groups are optionally substituted by another groupdescribed in the circumstance, the use of “independently optionally”means that each instance of a hydrogen atom on the group may besubstituted by another group, where the groups replacing each of thehydrogen atoms may be the same or different. Or for example, wheremultiple groups exist all of which can be selected from a set ofpossibilities, the use of “independently” means that each of the groupscan be selected from the set of possibilities separate from any othergroup, and the groups selected in the circumstance may be the same ordifferent.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which counter ions which may be used in pharmaceuticals.See, generally, S. M. Berge, et al., “Pharmaceutical Salts,” J. Pharm.Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts arethose that are pharmacologically effective and suitable for contact withthe tissues of subjects without undue toxicity, irritation, or allergicresponse. A compound described herein may possess a sufficiently acidicgroup, a sufficiently basic group, both types of functional groups, ormore than one of each type, and accordingly react with a number ofinorganic or organic bases, and inorganic and organic acids, to form apharmaceutically acceptable salt. Such salts include:

-   -   (1) acid addition salts, which can be obtained by reaction of        the free base of the parent compound with inorganic acids such        as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric        acid, sulfuric acid, and perchloric acid and the like, or with        organic acids such as acetic acid, oxalic acid, (D) or (L) malic        acid, maleic acid, methane sulfonic acid, ethanesulfonic acid,        p-toluenesulfonic acid, salicylic acid, tartaric acid, citric        acid, succinic acid or malonic acid and the like; or    -   (2) salts formed when an acidic proton present in the parent        compound either is replaced by a metal ion, e.g., an alkali        metal ion, an alkaline earth ion, or an aluminum ion; or        coordinates with an organic base such as ethanolamine,        diethanolamine, triethanolamine, trimethamine,        N-methylglucamine, and the like.

Pharmaceutically acceptable salts are well known to those skilled in theart, and any such pharmaceutically acceptable salt may be contemplatedin connection with the embodiments described herein. Examples ofpharmaceutically acceptable salts include sulfates, pyrosulfates,bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates,dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides,bromides, iodides, acetates, propionates, decanoates, caprylates,acrylates, formates, isobutyrates, caproates, heptanoates, propiolates,oxalates, malonates, succinates, suberates, sebacates, fumarates,maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates,chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates,methoxybenzoates, phthalates, sulfonates, methylsulfonates,propylsulfonates, besylates, xylenesulfonates, naphthalene-1-sulfonates,naphthalene-2-sulfonates, phenylacetates, phenylpropionates,phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates,tartrates, and mandelates. Lists of other suitable pharmaceuticallyacceptable salts are found in Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Company, Easton, Pa., 1985.

For a compound that contains a basic nitrogen, a pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamicacid, nitric acid, boric acid, phosphoric acid, and the like, or with anorganic acid, such as acetic acid, phenylacetic acid, propionic acid,stearic acid, lactic acid, ascorbic acid, maleic acid, hydroxymaleicacid, isethionic acid, succinic acid, valeric acid, fumaric acid,malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid,oleic acid, palmitic acid, lauric acid, a pyranosidyl acid, such asglucuronic acid or galacturonic acid, an alpha-hydroxy acid, such asmandelic acid, citric acid, or tartaric acid, an amino acid, such asaspartic acid or glutamic acid, an aromatic acid, such as benzoic acid,2-acetoxybenzoic acid, naphthoic acid, or cinnamic acid, a sulfonicacid, such as laurylsulfonic acid, p-toluenesulfonic acid,methanesulfonic acid, or ethanesulfonic acid, or any compatible mixtureof acids such as those given as examples herein, and any other acid andmixture thereof that are regarded as equivalents or acceptablesubstitutes in light of the ordinary level of skill in this technology.

The disclosure also relates to pharmaceutically acceptable prodrugs ofthe compounds and treatment methods employing such pharmaceuticallyacceptable prodrugs. The term “prodrug” means a precursor of adesignated compound that, following administration to a subject, yieldsthe compound in vivo via a chemical or physiological process such assolvolysis or enzymatic cleavage, or under physiological conditions(e.g., a prodrug on being brought to physiological pH is converted tothe compound in accordance with the present disclosure). A“pharmaceutically acceptable prodrug” is a prodrug that is non-toxic,biologically tolerable, and otherwise biologically suitable foradministration to the subject. Illustrative procedures for the selectionand preparation of suitable prodrug derivatives are described, forexample, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

Any formula depicted herein is intended to represent a compound of thatstructural formula as well as certain variations or forms. For example,a formula given herein is intended to include a racemic form, or one ormore enantiomeric, diastereomeric, or geometric isomers, or a mixturethereof. Additionally, any formula given herein is intended to referalso to a hydrate, solvate, or polymorph of such a compound, or amixture thereof. For example, it will be appreciated that compoundsdepicted by a structural formula containing the symbol “

” include both stereoisomers for the carbon atom to which the symbol “

” is attached, specifically both the bonds “

” and “

” are encompassed by the meaning of “

”.

Any formula given herein is also intended to represent unlabeled formsas well as isotopically labeled forms of the compounds. Isotopicallylabeled compounds have structures depicted by the formulas given hereinexcept that one or more atoms are replaced by an atom having a selectedatomic mass or mass number. Examples of isotopes that can beincorporated into compounds of the disclosure include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, andiodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F³⁶Cl, and ¹²⁵I, respectively. Such isotopically labelled compounds areuseful in metabolic studies (preferably with ¹⁴C), reaction kineticstudies (with, for example ²H or ³H), detection or imaging techniques[such as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT)] including drug or substrate tissuedistribution assays, or in radioactive treatment of patients. Further,substitution with heavier isotopes such as deuterium (i.e., ²H) mayafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements. Isotopically labeled compounds of this disclosure andprodrugs thereof can generally be prepared by carrying out theprocedures disclosed in the schemes or in the examples and preparationsdescribed below by substituting a readily available isotopically labeledreagent for a non-isotopically labeled reagent.

Any disubstituent referred to herein is meant to encompass the variousattachment possibilities when more than one of such possibilities areallowed. For example, reference to disubstituent -A-B-, where A≠B,refers herein to such disubstituent with A attached to a firstsubstituted member and B attached to a second substituted member, and italso refers to such disubstituent with A attached to the secondsubstituted member and B attached to the first substituted member.

It should be understood that some of the ring structures describedherein may be conjugated systems. Some of these conjugated systems mayform aromatic rings. The graphical representations described hereinshould be understood as exemplary graphical representations of thepossible other resonance structures or isomeric structures.

Example 1: Synthesis of Compounds

The synthesis of compounds according to the instant disclosure follows:

  Compound IDs From Instant Disclosure MBQ-1 (3a) MBQ-2 (3b) MBQ-3 (3c)MBQ-4 (3d) MBQ-5 MBQ-6 (4a) MBQ-7 (4b) MBQ-8 (4c) MBQ-9 (4d) MBQ-10 (4e)MBQ-11 (4f) MBQ-12 (7a) MBQ-13 (7b) MBQ-14 (7c) MBQ-15 (11a) MBQ-16(11b) HV-107 MBQ-17 (14a) MBQ-18 (14b) MBQ-19 (14c) MBQ-20 (14d) MBQ-21(14f) MBQ-22 (14e) MBQ-23 (14g) MBQ-24 (14h) 29 32 33 34 35 36 37 38 3940 41 HV-118  8

General Remarks

All experiments were carried out in pre-dried glassware (≥1 h, 80-90°C.) under a nitrogen atmosphere. Nuclear magnetic resonance (NMR)spectra were obtained using a 400 MHz Bruker Avance UltraShield™spectrometer. ¹H (400 MHz) and ¹³C (100 MHz) NMR were recorded in CDCl₃or DMSO-d₆, unless otherwise used, and the chemical shift were expressedin ppm relative to CDCl₃ (δ 7.26 for ¹H and δ 77.0 for ¹³C) or DMSO-d₆(δ 2.50 for ¹H and δ 39.5 for ¹³C) as the internal standard. ¹H NMR datais reported as position (δ), relative integral, multiplicity (s,singlet; d, doublet; t, triplet; q, quartet; dt, doublet of triplets;dd, doublet of doublets; dq, doublet of quartets; m, multiplet; br,broad peak), coupling constant (J, Hz), and the assignment of the atom.¹³C NMR data are reported as position (δ) and assignment of the atom.Microwave reactions were conducted in a CEM Discovery Microwave for DrugDiscovery, SP-1445.

Chemistry

Progress of the reaction was monitored via TLC analysis (General purposesilica gel on glass 5×20 cm with UV indicator, and visualized by UVfluorescent Spectroline E Series Ultraviolet lamps, in most casesfollowed by staining with I₂. The compounds were purified via columnchromatography over silica gel (70-230 mesh, 60 Å) with the appropriatesize column (24/40, 12 in.×0.5 in.) or (24/40, 12 in.×0.72 in.).

Synthesis of 2-substituted-nicotinamide Derivatives 3a-d Synthesis of2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide

In a 50 mL three-neck round-bottom flask was charged2-chloropyridine-3-carboxylic acid 1 (0.4726 g, 3.0 mmol), HOBT (0.4189g, 3.1 mmol), EDAC (0.5943 g, 3.1 mmol). The solution was dissolved inDMF (5 mL) and 3-Amino-9-ethylcarbazole 2 (0.6308 g, 3.0 mmol) wasadded. After 15 min, Et₃N (0.859 mL, 6.0 mmol) was added and the mixturestirred at room temperature for 16 h. After completion of the reaction,water was added (30 mL) and the product was extracted usingdichloromethane (30 mL). The organic layer was washed with brine anddried with Na₂SO₄, filtered and concentrated under reduced pressure. Thecrude oil was purified via column chromatography over silica gel and theproduct obtained as a white solid for the precursor2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (0.7565 g, 2.2 mmol,73%). TLC analysis in CH₂Cl₂-MeOH (9:1). Rf=0.61. ¹H NMR (400 MHz,CDCl₃) δ 1.46 (3H, t, J=7.3 Hz), 4.40 (2H, q, J=7.1 Hz), 7.43, (1H, t,J=8.6 Hz), 7.51 (11H, t, J=7.6 Hz), 7.64 (1H, d, J=2.3 Hz), 7.66 (1H, d,J=2.0 Hz), 8.13 (1H, d, J=7.3 Hz), 8.26 (1H, d, J=2.0 Hz), 8.28 (1H, d,J=1.8 Hz), 8.38 (1H, s), 8.47 (1H, d, J=1.8 Hz), 8.53 (1H, d, J=2.0 Hz),8.54 (1H, d, J=2.0 Hz); ¹³C NMR (100 MHz, CDCl₃) 14.1, 38.0, 108.9,113.6, 119.3, 121.1, 123.0, 123.2, 123.5, 126.4, 129.2, 132.0, 138.0,140.3, 140.8, 147.4, 151.4, 162.9.

Synthesis of2-(4-acetylpiperazin-1-yl)-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (3a)

In a 50 mL three neck round bottom flask equipped with a refluxcondenser was charged with2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (0.1749 g, 0.5 mmol),DIPEA (0.52 mL, 3.0 mmol) and CuI (0.03 g, 0.16 mmol) in 5 mL ofdioxane. After stirring for 5 min, 1-acetylpiperazine (0.077 g, 0.6mmol) was added and the solution was heated at 80° C. for 8 h. Aftercompletion of the reaction, the mixture was allowed to reach roomtemperature, water was added (30 mL) and the product was extracted withdichloromethane (3×30 mL). The combined organic phases were washed withbrine and dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The crude oil product was purified via columnchromatography over silica gel and the product 3a was obtained as ayellow solid (0.0506 g, 0.114 mmol, 23%). TLC analysis in CH₂Cl₂-MeOH(9:1), R_(f)=0.58. ¹H NMR (400 MHz, CDCl₃) δ 1.25 (1H, t, J=7.2 Hz),1.43 (3H, t, J=7.2 Hz), 2.09 (3H, s), 3.31 (4H, q, J=5.6 Hz), 3.64 (2H,t, J=5.2 Hz), 3.84 (2H, t, J=5.8 Hz), 4.12 (1H, q, J=7.2 Hz), 4.36 (2H,q, J=7.2 Hz), 7.22 (2H, m), 7.39 (2H, t, J=8.4 Hz), 7.47 (1H, t, J=8.0Hz), 7.54 (1H, d, J=2.0 Hz), 7.56 (1H, d, J=1.6 Hz), 8.11 (1H, d,J=7.6), 8.43 (1H, t, J=2.0 Hz), 8.45 (1H, d, J=2.0 Hz), 8.63 (1H, d,J=1.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 14.1, 21.3, 37.7, 41.5, 46.3,51.5, 108.6, 108.8, 112.2, 118.5, 118.9, 120.1, 120.77, 122.4, 122.72,123.4, 126.1, 129.8, 137.2, 140.4, 140.5, 150.0, 159.6, 163.1, 169.2.

Synthesis of N-(9-ethyl-9H-carbazol-3-yl)-2-morpholinonicotinamide (3b)

In a 50 mL three neck round bottom flask equipped with a refluxcondenser was charged with2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (0.1749 g, 0.5 mmol),DIPEA (0.52 mL, 3.0 mmol) and CuI (0.03 g, 0.16 mmol) in 5 mL ofdioxane. After stirring for 5 min, morpholine (0.052 mL, 0.6 mmol) wasadded and the solution was heated at 80° C. for 10 h. After completionof the reaction, the mixture was allowed to reach room temperature,water was added (30 mL) and the product was extracted withdichloromethane (3×30 mL). The combined organic phases were washed withbrine and dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The crude oil product was purified via columnchromatography over silica gel and the product 3b was obtained as awhite solid (0.0841 g, 0.21 mmol, 42%). TLC analysis in CH₂Cl₂-MeOH(9:1) R_(f)=0.79. ¹H NMR (400 MHz, CDCl3) δ 1.46 (3H, t, J=7.2 Hz), 3.32(4H, t, J=4.4 Hz), 3.95 (4H, t, J=4.4 Hz), 4.37 (2H, q, J=7.2 Hz), 7.24(2H, m), 7.41 (1H, d, J=7.2 Hz), 7.48 (1H, t, J=7.2 Hz), 7.63 (1H, d,J=1.6 Hz), 7.66 (1H, d, J=1.6 Hz), 8.14 (1H, d, J=8.0 Hz), 8.46 (1H, d,J=4.4 Hz), 8.48 (1H, d, J=1.6 Hz), 8.67 (1H, s); ¹³C NMR (100 MHz,CDCl3) δ 14.1, 37.9, 52.1, 67.4, 108.8, 109.0, 112.3, 118.8, 119.1,120.2, 121.0, 122.6, 123.0, 123.5, 126.3, 130.2, 137.4, 140.7, 140.7,150.3, 160.2, 163.3.

Synthesis ofN-(9-ethyl-9H-carbazol-3-yl)-2-(4-phenylpiperazin-1-yl)nicotinamide (3c)

In a 50 mL three neck round bottom flask equipped with a refluxcondenser was charged with2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (0.1574 g, 0.45 mmol),DIPEA (0.52 mL, 3.0 mmol) and CuI (0.03 g, 0.16 mmol) in 5 mL ofdioxane. After stirring for 5 min, 1-phenylpiperazine (0.070 mL, 0.46mmol) was added and the solution was heated at 80° C. for 10 h. Aftercompletion of the reaction, the mixture was allowed to reach roomtemperature, water was added (30 mL) and the product was extracted withdichloromethane (3×30 mL). The combined organic phases were washed withbrine and dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The crude oil product was purified via columnchromatography over silica gel and the product 3c was obtained as abrown solid (0.0476 g, 0.10 mmol, 22%). TLC analysis in CH₂Cl₂-MeOH(9:1) R_(f)=1.0. ¹H NMR (400 MHz, CDCl₃) δ 1.43 (3H, t, J=7.2 Hz), 4.36(2H, q, J=7.2 Hz), 7.29 (t, J=8.8 Hz), 7.39 (t, J=8.6 Hz), 7.47 (t,J=7.0 Hz), 7.54 (d, J=2.3 Hz), 7.56 (d, J=2.3 Hz), 8.09 (d, J=7.7 Hz),8.47 (d, J=1.9 Hz), 8.49 (d, J=1.9), 8.51 (d, J=1.8 Hz), 8.52 (d, J=1.8Hz), 8.73 (d, J=1.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 13.8, 37.6, 49.7,51.7, 108.5, 108.8, 112.2, 116.4, 118.5, 118.8, 120.0, 120.8, 122.5,122.8, 123.3, 125.9, 129.3, 130.0, 137.1, 140.4, 140.5, 150.0, 163.1.

Synthesis ofN-(9-ethyl-(H-carbazol-3-yl)-2-((3-morpholinopropyl)amino)nicotinamide(3d)

In a 50 mL three neck round bottom flask equipped with a refluxcondenser was charged with2-chloro-N-(9-ethyl-9H-carbazol-3-yl)nicotinamide (0.350 g, 1.00 mmol),DIPEA (0.52 mL, 3.0 mmol) and CuI (0.03 g, 0.16 mmol) in 5 mL ofdioxane. After stirring for 5 min, 3-morpholinopropylamine (0.16 mL, 1.1mmol) was added in one portion and the solution was heated at 80° C. for10 h. After completion of the reaction, the mixture was allowed to reachroom temperature, water was added (30 mL) and the product was extractedwith dichloromethane (3×30 mL). The combined organic phases were washedwith brine and dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The crude oil product was purified via columnchromatography over silica gel and the product 3d was obtained as awhite solid (0.0985 g, 0.105 mmol, 11%). TLC analysis in CH₂Cl₂-MeOH(9:1), R_(f)=0.29. ¹H NMR (400 MHz, CDCl₃) δ 1.44 (3H, t, J=7.2 Hz),1.85 (2H, m), 2.47 (5H, t, J=7.2 Hz), 3.57 (2H, q, J=6.8 Hz), 3.72 (4H,t, J=4.6 Hz), 4.39 (2H, q, J=7.2 Hz), 6.56 (1H, q, J=5.1 Hz), 7.24 (1H,t, J=7.3 Hz), 7.41 (1H, d, J=5.8 Hz), 7.42 (1H, d, J=5.1 Hz), 7.48 (1H,t J=6.6 Hz), 7.53 (1H, d, J=1.8 Hz), 7.55 (1H, d, J=2.3 Hz), 7.76 (1H,d, J=2.0 Hz), 7.78 (1H, d, J=1.0 Hz), 7.84 (1H, s), 8.10 (1H, d, J=8.3Hz), 8.27 (1H, d, J=2.3 Hz), 8.28 (1H, d, J=1.8 Hz), ¹³C NMR (100 MHz,CDCl₃) δ 13.8, 26.4, 37.6, 39.5, 53.8, 56.8, 66.9, 108.7, 110.4, 113.9,118.9, 120.4, 120.7, 122.6, 123.2, 126.0, 129.0, 135.1, 137.6, 140.5,157.1, 158.2, 166.9.

Synthesis of 2-substituted-nicotinamide Derivatives 4a-f Synthesis of2-(9-ethyl-9H-carbazol-3-ylamino)-N-(3-norpholinopropyl)nicotinamide(4a)

Method A. Step (i) A microwave tube was charged with 2-chloronicotinicacid 1 (0.1576 g, 1.0 mmol), 9-ethyl-9H-carbazol-3-amine 2 (0.6318 g,3.0 mmol), DIPEA (0.522 mL, 3.0 mmol), and water (1.5 mL). The solutionwas heated at 140° C. for 5 h under microwave conditions (Power levelset to 200 W; Caution! Pressure may develop). After completion of thereaction, the mixture was allowed to cool to room temperature andtransferred to a separatory funnel and diluted with dichloromethane (30mL). The solution was washed with water (3×20 mL). The organic phase wasseparated and dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The crude oil product was purified via columnchromatography over silica gel and the product intermediate2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid was obtained as awhite solid (0.1286 g, 0.39 mmol, 39%). ¹H NMR (400 MHz, CDCl₃) δ 1.32(t, 3H, J=7.18 Hz), 4.44 (q, 2H, J=6.98 Hz), 6.81 (q, 1H, J=4.8 Hz),7.17 (t, 1H, J=7.3 Hz), 7.44 (t, 1H, J=8.6 Hz), 7.58 (q, 2H, J=5.1 Hz),7.66 (d, 1H, J=1.8 Hz), 7.68 (d, 1H, J=1.5 Hz), 8.13 (d, 1H, J=7.8 Hz),8.23 (d, 1H, J=1.8 Hz), 8.25 (d, 1H, J=2.0 Hz), 8.35 (d, 1H, J=1.8 Hz),8.38 (d, 1H, J=2.0 Hz), 8.42 (s, 1H), 10.37 (s, 1H); ¹³C NMR (100 MHz,CDCl₃) δ 13.7, 37.0, 106.8, 108.9, 109.0, 113.0, 113.1, 118.4, 120.4,121.1, 122.1, 122.1, 125.2, 131.5, 136.1, 139.9, 140.4, 153.0, 156.4,169.2. Step (ii) In a 50 mL three neck round bottom flask equipped witha reflux condenser was charged with2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid (0.0634 g, 0.2 mmol),HOBt (0.0405 g, 0.2 mmol) and EDAC (0.0575 g, 0.3 mmol) in DMF (3 mL).To the solution was added 3-morpholinopropylamine (0.031 mL, 0.21 mmol).The solution is stirred at room temperature for 8 h until completed byTLC analysis. The reaction mixture is washed with water (10 mL) andextracted with ethyl acetate (3×20 mL). The combined organic phases arewashed with brine and dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude oil was purified viacolumn chromatography over silica gel and the product 4a was obtained asa white solid (0.0458 g, 0.1 mmol, 50%). TLC analysis in CH₂Cl₂-MeOH(9:1), R_(f)=0.35. ¹H NMR (400 MHz, CDCl₃) δ 1.43 (3H, t, J=7.6 Hz),1.83 (3H, t, J=5.8 Hz), 2.54 (3H, s), 2.60 (2H, t, J=5.3 Hz), 3.60 (2H,q, J=5.8 Hz), 3.74 (4H, t, J=4.6 Hz), 4.37 (2H, q, J=7.3 Hz), 6.66 (1H,q, J=4.5 Hz), 7.19 (1H, t, J=7.8 Hz), 7.36 (1H, d, J=4.0 Hz), 7.39 (1H,d, J=3.5 Hz), 7.44 (1H, t, J=7.1 Hz), 7.69 (1H, d, J=1.5 Hz), 7.71 (1H,d, J=2.0 Hz), 7.74 (1H, d, J=1.5 Hz), 7.76 (1H, d, J=1.5 Hz), 8.08 (1H,d, J=7.8 Hz), 8.31 (1H, d, J=1.8 Hz), 8.32 (1H, d, J=1.7 Hz), 8.35 (1H,d, J=1.7 Hz), 10.53 (1H, s); ¹³C NMR (100 MHz, CDCl₃) δ 13.9, 23.65,4.56, 40.9, 53.9, 59.0, 67.0, 108.4, 111.8, 113.7, 118.3, 120.6, 121.2,125.40, 131.90, 135.4, 136.7, 140.4, 151.6, 156.3, 168.4.

Synthesis of tert-butyl4-(2-(2-(9-ethyl-9H-carbazol-3-yl)amino)nicotinamido)ethyl)piperazine-1-carboxylate(4b)

Method B. (i) In a 100 mL three-neck round-bottom flask was charged2-chloronicotinic acid 1 (0.3151 g, 2.0 mmol), HOBt (0.4054 g, 3.0mmol), and EDAC (0.5751 g, 3.0 mmol) were dissolved in CH₂Cl₂ (10 mL).After stirring for 10 min, 4-(2-aminoethyl)-1-boc-piperazine (0.4586 mL,2.0 mmol) was added followed by Et₃N (0.5733 mL, 4.0 mmol) and thereaction mixture stirred at room temperature for 16 h. After completionof the reaction, the solution was washed with water (30 mL), and theproduct was extracted with CH₂Cl₂ (3×20 mL). The combined organic phaseswere washed with brine, and dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure. The concentrated crude oiltert-butyl 4-(2-(2-chloronicotinamido)ethyl)piperazine-1-carboxylateintermediate was obtained (0.63 g, 1.71 mmol, 86%). The product was usedin the next step without further purification. Step (ii) In a 100 mLthree neck round bottom flask equipped with a stirring bar and refluxcondenser, tert-butyl 4-(2-(2chloronicotinamido)ethyl)piperazine-1-carboxylate (0.630 g, 1.71 mmol)and 9-ethyl-9H-carbazol-3-amine (0.0361 g, 1.72 mmol) were dissolved in5 mL of DMSO. To the solution, CuI (0.0651 g, 0.3 mmol) and Cs₂CO₃ (1.11g, 3.42 mmol) were added and heated at 90° C. for 24 hr. After thereaction was complete (analyzed by TLC), the mixture was allowed toreach room temperature. The mixture was washed with water (30 mL), andextracted with CH₂Cl₂ (3×30 mL). The combined organic phases were washedwith brine and dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure. The crude oil was purified via columnchromatography over silica gel and the product 4b was obtained as awhite solid (0.08 g, 0.15 mmol, 9%). TLC analysis in CH₂Cl₂-MeOH (9:1),R_(f)=0.44. ¹H NMR (400 MHz, CDCl₃) δ 1.26 (4H, t, J=7.1 Hz), 1.42 (3H,t, J=7.3 Hz), 1.46 (9H, s), 1.65 (3H, s), 2.04 (2H, s), 2.48 (3H, t,J=4.6 Hz), 2.65 (2H, t, J=5.9 Hz), 3.47 (4H, t, J=5.3 Hz), 3.56 (2H, q,J=5.6 Hz), 4.11 (1H, q, J=7.1 Hz), 4.34 (2H, q, J=7.6 Hz) 6.64 (1H, q,J=4.8 Hz), 7.19 (1H, t, J=6.3 Hz), 7.26 (1H, s,), 7.35 (1H, d, J=4.3Hz), 7.39 (1H, d, J=3.8 Hz), 7.43 (1H, t, J=7.6 Hz), 7.65 (1H, d, J=1.8Hz), 7.67 (1H, t, J=2.3 Hz), 7.69 (1H, d, J=2.0 Hz), 8.08 (1H, d, 7.6Hz), 8.31 (1H, d, J=1.5 Hz), 8.32 (1H, d, J=1.8 Hz), 8.33 (1H, d, J=2.0Hz), 10.40 (1H, s); ¹³C NMR (100 MHz, CDCl₃) δ 13.8, 37.5, 42.2, 44.8,45.5, 66.6, 108.3, 112.8, 115.3, 118.3, 120.5, 120.7, 122.8, 123.2,125.4, 131.6, 132.8, 136.5, 132.0, 140.3, 147.6, 167.8.

Synthesis ofN-(3-(1H-imidazol-1-yl)propyl)-2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinamide(4c)

Method A. Step (i), the product intermediate2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid was prepared as inprocedure for compound 4a. Step (ii) In a 100 mL three-neck round-bottomflask was charged with 2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid(0.285 g, 0.9 mmol), HOBt (0.1351 g, 1.0 mmol), and EDAC (0.1917 g, 1.0mmol) dissolved in CH₂Cl₂ (10 mL). To the solution,1-(3-aminopropyl)imidazole (0.1190 mL, 1.0 mmol) was added. After 15 minof stirring, Et₃N (0.286 mL, 2.0 mmol) was added and the reactionmixture was stirred at room temperature overnight. After the reactionwas complete (analyzed by TLC), the mixture was washed with water (30mL), and extracted with CH₂Cl₂ (3×20 mL). The combined organic phaseswere washed with brine and dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure. The crude was purified via columnchromatography over silica gel and the product 4c was obtained as awhite solid (0.0324 g, 0.074 mmol, 9%). TLC analysis in CH₂Cl₂-MeOH(9:1), R_(f)=0.50. ¹H NMR (400 MHz, CDCl₃) δ 1.26 (4H, s), 1.42 (3H, t,J=7.2 Hz), 2.12 (5H, t, J=6.0 Hz), 3.44 (2H, q, J=6.8 Hz), 4.05 (2H, t,J=6.8 Hz), 4.35 (2H, q, J=7.2 Hz), 6.60 (1H, q, J=4.8 Hz), 6.93 (2H, s),7.06 (1H, s), 7.16 (1H, t, J=3.2 Hz), 7.37 (1H, t, J=8.4 Hz), 7.40 (11H,t, J=8.8 Hz), 7.51 (1H, s), 7.67 (2H, t, J=8.8 Hz), 8.06 (1H, d, J=7.2Hz), 8.28 (1H, d, J=4.4 Hz), 8.32 (1H, s), 10.36 (1H, s); ¹³C NMR (100MHz, CDCl₃) δ 13.8, 29.7, 30.9, 37.2, 37.6, 44.8, 108.4, 110.1, 112.0,113.9, 118.4, 118.9, 120.6, 121.4, 123.0, 123.1, 125.5, 129.5, 131.7,135.6, 136.8, 137.1, 140.4, 151.8, 156.3, 168.5.

Synthesis ofN-(4-Diethylamino-1-methyl-butyl)-2-(9-ethyl-9H-carbazol-3-ylamino)-nicotinamide(4d)

Method A. Step (i), the product intermediate2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid was prepared as inprocedure for compound 4a. Step (ii) In a 100 mL three-neck round-bottomflask was charged with 2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid(0.9941 g, 3.0 mmol), HOBt (0.4054 g, 3.0 mmol), and EDAC (0.5751 g, 3.0mmol) dissolved in CH₂Cl₂ (10 mL). After 15 min of stirring,2-amino-5-diethylaminopentane (0.58 g, 3.0 mmol) was added followed byEt₃N (0.43 mL, 3.0 mmol). The reaction mixture was stirred at roomtemperature for 16 h. After the reaction was complete (analysis by TLC),water (30 mL) was added, and the product extracted with CH₂Cl₂ (3×20mL). The combined organic phases were washed with brine and dried overanhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Thecrude was purified via column chromatography over silica gel and theproduct 4c was obtained as a white solid (0.500 g, 1.0 mmol, 33.3%). TLCanalysis in CH₂Cl₂-MeOH (9:1), R_(f)=0.42. ¹H NMR (400 MHz, CDCl₃) δ1.29 (10H, m), 1.42 (3H, t, J=7.2 Hz), 3.02 (4H, q, J=6.4 Hz), 4.38 (2H,q. J=7.2 Hz), 6.67 (1H, q, J=4.8 Hz), 7.16 (1H, t, J=6.8 Hz), 7.38 (2H,t, J=6.4 Hz), 7.46 (1H, t, J=8.4 Hz), 7.68 (1H, d, J=2.4 Hz), 7.71 (1H,d, J=2.0 Hz), 8.07 (1H, d, J=7.6 Hz), 8.12 (1H, d, J=7.2 Hz), 8.28 (1H,d, J=4.0 Hz), 8.32 (1H, d, J=1.6 Hz), 10.56 (1H, s); ¹³C NMR (100 MHz,CDCl₃) δ 9.0, 14.2, 21.8, 33.6, 37.9, 45.6, 47.2, 52.5, 108.7, 110.7,112.7, 113.9, 118.7, 120.9, 121.5, 123.3, 125.7, 132.4, 136.9, 140.7,151.8, 156.6, 168.7.

Synthesis ofN-(2-(diethylamino)ethyl)-2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinamide(4e)

Method A. Step (i), the product intermediate2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid was prepared as inprocedure for compound 4a. Step (ii) In a 100 mL three-neck round-bottomflask was charged with 2-((9-ethyl-9H-carbazol-3-yl)amino)nicotinic acid(0.9941 g, 3.0 mmol), HOBt (0.4054 g, 3.0 mmol), and EDAC (0.5751 g, 3.0mmol) dissolved in CH₂Cl₂ (10 mL). After 15 min of stirring,N,N-diethylethylenediamine (0.42 mL, 3.0 mmol) and Et₃N (0.42 mL, 3.0mmol) were added. The reaction mixture was stirred at room temperaturefor 16 h. After the reaction was complete (analysis by TLC), water (30mL) was added, and the product extracted with CH₂C₁₂ (3×20 mL). Thecombined organic phases were washed with brine and dried over anhydrousNa₂SO₄, filtered, and concentrated under reduced pressure. The crude waspurified via column chromatography over silica gel and the product (4e)was obtained as a white solid (0.7984 g, 1.8 mmol, 60%). TLC analysis inCH₂Cl₂-MeOH (9:1), R_(f)=0.67. ¹H NMR (400 MHz, CDCl₃) δ 1.29 (3H, t,J=7.3 Hz), 1.42 (3H, t, J=7.3 Hz), 3.04 (q, J=6.8 Hz), 3.14 (t, J=5.0Hz), 3.78 (q, J=5.56 Hz), 4.35 (q, J=7.1 Hz), 7.18 (t, J=7.6), 7.36 (d,J=4.8 Hz), 7.39 (d, J=3.8 Hz), 7.44 (t, J=7.1 Hz), 7.68 (d, J=2.0 Hz),7.71 (d, J=2.78 Hz), 8.06 (d, J=7.6 Hz), 8.31 (d, J=1.8 Hz); ¹³C NMR(100 MHz, CDCl₃) δ 8.6, 13.5, 35.4, 37.2, 47.2, 51.6, 108.0, 108.0,109.3, 112.1, 113.6, 118.0, 120.3, 121.1, 122.7, 122.8, 125.1, 131.6,136.4, 140.0, 151.5, 156.1, 168.7.

Synthesis of2-((9-ethyl-9H-carbazol-3-yl)amino)-N-(3,4,5-trimethoxyphenyl)nicotinamide(4f)

Method B. Step (i) In a 100 mL three-neck round-bottom flask was charged2-chloronicotinic acid 1 (0.1576 g, 1.0 mmol), HOBt (0.2027 g, 1.5mmol), and EDAC (0.2876 g, 1.5 mmol) dissolved in CH₂Cl₂ (10 mL). Themixture was stirred for 10 min, then 3,4,5-trimethoxyaniline (0.1832 g,1.0 mmol) and Et₃N (0.3 mL, 2.0 mmol) were added. The mixture wasstirred at room temperature for 16 h. After the reaction was complete(analyzed by TLC), the solution was washed with water (30 mL), and theproduct was extracted with CH₂Cl₂ (3×20 mL). The combined organic phaseswere washed with brine and dried over anhydrous Na₂SO₄, filtered, andconcentrated under reduced pressure. The crude oil was purified viacolumn chromatography over silica gel and the product2-chloro-N-(3,4,5-trimethoxyphenyl)nicotinamide was obtained as a whitesolid (0.2828 g, 0.8762 mmol, 87.6%). TLC analysis in ethylacetate-hexane (1:1), R_(f)=0.34. ¹H NMR (400 MHz, CDCl₃) δ 3.88 (5H,s), 3.83 (3H, s), 6.94 (2H, s), 7.26 (1H, s), 7.39 (1H, q, J=4.76 Hz)8.18 (1H, d, J=1.98 Hz), 8.50 (11H, d, J=1.97 Hz), 8.51 (1H, d, J=1.95Hz): ¹³C NMR (100 MHz, CDCl₃) δ 14.66, 56.22, 60.98, 98.00, 122.97,131.38, 133.38, 133.21, 135.51, 139.93, 146.92, 151.36, 153.51, 162.50.Step (ii) In a 100 mL three neck round bottom flask equipped with astirring bar and reflux condenser,2-chloro-N-(3,4,5-trimethoxyphenyl)nicotinamide (0.2828 g, 0.88 mmol)and 9-ethyl-9H-carbazol-3-amine 2 (0.1517 g, 0.7214 mmol) were dissolvedin DMSO (3 mL). To the solution, CuI (40 mg, 0.21 mmol, 25%) and Cs₂CO₃(1.0 g, 3.0 mmol) were added, and heated at 90° C. for 24 h. After thereaction was complete (analyzed by TLC), the mixture was allowed toreach room temperature. The mixture was washed with water (30 mL), andextracted with CH₂Cl₂ (3×30 mL). The combined organic phases were washedwith brine and dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure. The crude oil was purified via columnchromatography over silica gel and the product 4f was obtained as awhite solid (0.0324 g, 0.0652 mmol, 8%). TLC analysis in ethylacetate-hexane (1:1), R_(f)=0.35. ¹H NMR (400 MHz, CDCl₃) δ 1.35 (10H,t, J=7.3 Hz), 1.49 (3H, t, J=7.6 Hz), 1.62 (7H, s), 2.08 (3H, s), 3.87(8H, s), 4.17 (2H, q, J=7.3 Hz), 4.43 (1H, q, J=7.3 Hz), 5.51 (1H, s),6.65 (1H, s), 6.95 (11H, q, J=4.6 Hz), 7.30 (1H, s), 7.47 (1H, d, J=8.3Hz), 7.51 (2H, d, J=6.32 Hz), 8.09 (1H, d, J=9.4 Hz), 8.36 (1H, q, J=2.5Hz), 8.41 (1H, d, J=2.0 Hz), 8.43 (1H, d, J=1.5 Hz); ¹³C NMR (100 MHz,CDCl₃) δ 13.9, 37.7, 56.3, 60.9, 68.4, 103.2, 108.7, 109.2, 112.9,115.8, 117.7, 119.0, 120.6, 122.6, 123.7, 124.0, 126.1, 133.7, 136.0,137.1, 138.2, 138.5, 140.5, 153.1, 153.6, 157.7, 163.0.

Synthesis of 2, 3-diamino-substituted Pyridine Derivatives 7a-c and11a-b Synthesis of(9-Ethyl-9H-carbazol-3-yl)-(3-nitro-pyridin-2-yl)-amine (6)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser, 2-chloro-3-nitropyridine 5 (0.31708 g, 2 mmol),9-ethyl-9H-carbazol-3-amine 2 (0.42056 g, 2 mmol), and Et₃N (0.3582 mL,2.5 mmol) were dissolved in THF (3 mL). The reaction mixture is refluxedfor 2 h. After the reaction was complete (analyzed by TLC), the mixturewas allowed to reach room temperature. The mixture was washed with water(30 mL), and extracted with ethyl acetate (3×30 mL). The combinedorganic phases were washed with brine and dried over anhydrous Na₂SO₄,filtered, and concentrated under reduced pressure. The crude waspurified via column chromatography over silica gel and the product(9-Ethyl-9H-carbazol-3-yl)-(3-nitro-pyridin-2-yl)-amine 6 was obtainedas a brown solid (0.16 g, 0.48 mmol, 24.1%). TLC analysis in CH₂C₁₂-MeOH(9:1), R_(f)=0.71. ¹H NMR (400 MHz, DMSO-d₆) δ 1.34 (3H, t, J=7.1 Hz),4.48 (2H, q, J=6.6 Hz), 6.94 (1H, q, J=4.4 Hz), 7.20 (1H, t, J=7.6 Hz),7.47 (1H, t, J=7.2 Hz), 7.63 (2H, s), 8.15 (1H, d, J=8.0 Hz), 8.34 (s,1H), 8.49 (1H, d, J=3.2 Hz), 8.57 (1H, d, J=8.4 Hz), 10.13 (1H, s); ¹³CNMR (100 MHz, DMSO-d₆) δ 14.2, 37.5, 109.4, 109.7, 114.2, 116.6, 119.2,120.9, 122.5, 123.7, 126.3, 128.6, 130.3, 136.0, 137.6, 140.5, 151.1,156.1.

Synthesis of N2-(9-Ethyl-9H-carbazol-3-yl)-pyridine-2,3-diamine (6a)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser,9-ethyl-N-(3-nitropyridin-2-yl)-9H-carbazol-3-amine 6 (0.16 g, 0.48mmol) was dissolved in EtOH (25 mL) and water (25 mL). After stirringfor 10 min, SnCl₂.2H₂O (0.27 mL, 1.44 mmol) and HCl (0.5 mL) were added,and the reaction mixture was refluxed for 5 h. After reaction completion(analysis by TLC), the mixture was allowed to reach room temperature andwashed with 1M KOH. The layers were separated and the aqueous layer wasfurther extracted with ethyl acetate (3×30 mL). The combined organicphases were washed with brine and dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure. The crude oil productN²-(9-Ethyl-9H-carbazol-3-yl)-pyridine-2,3-diamine 6a was utilizedwithout further purification. TLC analysis in CH₂Cl₂-MeOH (9:1),R_(f)=0.43. ¹H NMR (400 MHz, DMSO-d₆) δ 1.32 (3H, t, J=7.03 Hz), 4.40(2H, q, J=6.97 Hz), 5.02 (2H, s), 6.56 (1H, dd, J=4.92, 7.22 Hz), 6.88(1H, d, J=7.45 Hz), 7.14 (1H, t, J=7.40 Hz), 7.41 (1H, t, J=7.37 Hz),7.49 (1H, d, J=8.19 Hz), 7.55 (1H, d, J=8.14 Hz), 7.66 (1H, d, J=12.57Hz), 8.03 (1H, d, J=7.65 Hz), 8.36 (1H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ13.5, 36.7, 108.4, 108.7, 110.5, 114.4, 117.9, 118.8, 119.7, 119.8,121.7, 122.0, 125.1, 130.1, 134.1, 134.3, 134.8, 139.6, 144.7.

Synthesis ofN-[2-(9-Ethyl-9H-carbazol-3-ylamino)-pyridin-3-yl]-3-piperidin-1-yl-propionamide(7a)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser, N²-(9-ethyl-9H-carbazol-3-yl)pyridine-2,3-diamine6a (0.160 g, 0.53 mmol), 1-piperidinepropionic acid (0.1570 g, 1.0mmol), HOBt (0.2027 g, 1.5 mmol), and EDAC (0.2876 g, 1.5 mmol)dissolved in CH₂Cl₂ (10 mL). After stirring for 30 minutes, Et₃N (0.3mL) was added and the reaction mixture stirred for 24 hr. After thereaction was complete (analyzed by TLC), the mixture was allowed toreach room temperature. The mixture was washed with water (30 mL), andextracted with CH₂Cl₂ (3×30 mL). The combined organic phases were washedwith brine and dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure. The crude was purified via column chromatographyover silica gel and the product 7a was obtained (0.1630 g, 0.37 mmol,65%) as a yellow solid. TLC analysis in CH₂Cl₂-MeOH (9:1), R_(f)=0.53.¹H NMR (400 MHz, CDCl₃) δ 1.41 (3H, t, J=7.1 Hz), 1.67 (4H, t, J=6.1Hz), 2.76 (2H, d, J=4.0 Hz), 2.85 (2H, d, J=4.6 Hz), 4.33 (2H, q, J=7.3Hz), 6.75 (1H, q, J=5.1 Hz), 7.17 (1H, t, J=7.3 Hz), 7.35 (3H, t, J=9.1Hz), 7.43 (1H, t, J=6.8 Hz), 7.53 (1H, d, J=8.1 Hz), 7.69 (1H, d, J=8.1Hz), 8.05 (1H, d, J=10.3 Hz), 8.08 (1H, d, J=4.3 Hz); ¹³C NMR (400 MHz,CDCl₃) δ 13.9, 23.4, 25.1, 32.2, 37.6, 53.9, 54.3, 108.3, 108.5, 113.5,114.5, 118.3, 120.6, 121.2, 122.9, 123.2, 125.4, 131.7, 132.6, 136.7,140.3, 144.7, 150.5, 171.1.

Synthesis ofN-(2-((9-ethyl-9H-carbazol-3-yl)amino)pyridin-3-yl)nicotinamide (7b)

Step (i) The N²-(9-Ethyl-9H-carbazol-3-yl)-pyridine-2,3-diamine wasprepared following procedure as in Step (i) 7a. Step (ii) In a 100 mLthree neck round bottom flask equipped with a stirring bar and refluxcondenser, N²-(9-ethyl-9H-carbazol-3-yl)pyridine-2,3-diamine (0.21 g,0.70 mmol) and nicotinoyl chloride hydrochloride (0.1780 g, 1.0 mmol)dissolved in THF (5 mL). To the mixture sodium bicarbonate (0.13 mL, 1.6mL) was added. After stirring for 30 minutes, Et₃N (0.1 mL) was addedand the reaction mixture refluxed for 24 hr. After the reaction wascomplete (analyzed by TLC), the mixture was allowed to reach roomtemperature. The mixture was washed with water (30 mL), and extractedwith CH₂Cl₂ (3×30 mL). The combined organic phases were washed withbrine and dried over anhydrous Na₂SO₄, filtered, and concentrated underreduced pressure. The crude oil was purified via column chromatographyover silica gel and the product 7b was obtained (0.240 g, 0.44 mmol,63%) as a yellow solid. TLC analysis in ethyl acetate-hexane (1:3),R_(f)=0.60. ¹H NMR (400 MHz, DMSO-d₆) δ 1.31 (3H, t, J=7.3 Hz) 4.43 (2H,q, J=7.3 Hz), 6.81 (1H, q, J=4.1 Hz), 7.15 (1H, t, J=7.8 Hz), 7.42 (1H,t, J=7.6 Hz), 7.5 (1 h, t, J=7.1 Hz), 7.67 (1H, d, J=8.6 Hz), 8.04 (1H,d, J=7.1 Hz), 8.27 (1H, s), 8.32 (1H, s), 8.41 (1H, d, J=8.6 Hz), 8.78(1H, d, J=3.3 Hz), 9.23 (1H, s), 10.03 (1H, s); ¹³C NMR (100 MHz,DMSO-d₆) δ 13.8, 36.7, 108.8, 112.5, 113.2, 119.0, 119.9, 121.7, 123.1,125.2, 129.9, 132.8, 134.7, 135.5, 139.7, 144.6, 148.9, 151.8, 164.6.

Synthesis of2-(2-(9-ethyl-9H-carbazol-4-yl)aminopyridin-3-yl)amino)-1-morpholinoethanone(7c)

The N²-(9-Ethyl-9H-carbazol-3-yl)-pyridine-2,3-diamine was preparedfollowing procedure as in Step (i) 7a. Step (ii) In a 100 mL three neckround bottom flask equipped with a stirring bar and reflux condenser,N²-(9-ethyl-9H-carbazol-3-yl)pyridine-2,3-diamine 6a,4-(chloroacetyl)morpholine (0.08 mL, 0.6 mmol) and Et₃N (0.07 mL) weredissolved in THF (5 mL). The reaction mixture was refluxed for 24 huntil the reaction was complete as indicated by TLC analysis. Themixture was washed with water (30 mL), and extracted with CH₂Cl₂ (3×30mL). The combined organic phases were washed with brine and dried overanhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Thecrude was purified via column chromatography over silica gel and theproduct 7c was obtained as a white solid (0.18 g, 0.42 mmol, 70%). TLCanalysis in CH₂Cl₂-MeOH (9:1), R_(f)=0.34. ¹H NMR (400 MHz, CDCl₃) δ1.45 (3H, t, J=7.1 Hz), 3.49 (2H, t, J=7.1 Hz) 3.72 (4H, t, J=6.0 Hz),3.92 (2H, s), 4.38 (2H, q, J=6.8 Hz), 6.77 (1H, t, J=7.3 Hz), 6.90 (1H,d, J=6.3 Hz), 7.22 (1H, t, J=7.21 Hz), 7.29 (1H, s), 7.39 (1H, d, J=4.3Hz), 7.41 (1H, d, J=4.3 Hz), 7.46 (1H, t, J=6.8 Hz), 7.52 (1H, d, J=8.6Hz), 8.05 (2H, d, J=8.1 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 13.9, 14.2,21.0, 28.4, 29.7, 36.1, 37.6, 52.7, 56.3, 60.4, 108.4. 110.4, 112.0,113.9, 118.4, 120.6, 121.4, 123.0, 125.4, 131.7, 135.3, 136.8, 140.3,151.7, 154.7, 156.3, 168.3.

General Method for the Synthesis of 2-amino-3-nitropyridine derivatives(8a-b)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser, 2-chloro-3-nitropyridine 5, 1 equiv. of primary orsecondary amine, and Et₃N (1 equiv.) were dissolved in THE (3 mL). Thereaction mixture is refluxed at 65° C. for 2 h. After the reaction wascomplete (analyzed by TLC), the mixture was allowed to reach roomtemperature. The mixture was washed with water (30 mL), and extractedwith ethyl acetate (3×30 mL). The combined organic phases were washedwith brine and dried over anhydrous Na₂SO₄, filtered, and concentratedunder reduced pressure. The crude oil was purified via columnchromatography with silica gel to obtain the appropriate2-amino-3-nitropyridine derivative 8a or 8b.

(3-Morpholin-4-yl-propyl)-(3-nitro-pyridin-2-yl)-amine (8a)

¹H NMR (400 MHz, CDCl₃) δ 1.86 (2H, quint, J=6.66 Hz), 2.48 (6H, m),3.69 (2H, t, J=6.51 Hz), 3.75 (4H, t, J=4.50 Hz), 6.61 (1H, t, J=5.70Hz), 8.40 (1H, d, J=7.10 Hz), 8.62 (1H, br); ¹³C NMR (100 MHz, CDCl₃) δ25.5, 40.3, 53.9, 57.2, 66.7, 111.4, 128.0, 135.2, 152.6, 155.7.

4-(3-Nitro-pyridin-2-yl)-morpholine (8b)

¹H NMR (400 MHz, CDCl₃) δ 3.45 (4H, t, J=4.48 Hz), 3.79 (4H, t, J=4.33Hz), 6.78 (1H, dd, J=4.48, 7.86 Hz), 8.12 (1H, d, J=7.98 Hz), 8.33 (1H,d, J=4.38 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 48.4, 66.6, 113.8, 133.2,135.6, 151.8, 152.7.

General Method for the Synthesis of 2,3-diaminopyridine Derivatives(9a-b)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser, 2-amino-3-nitropyridine derivative 8a or 8b wasdissolved in EtOH (25 mL) and water (25 mL). After stirring for 10 min,SnCl₂.2H₂O (1.5 equiv) and HCl were added, and the reaction mixture wasrefluxed at 80° C. for 5 h. After reaction completion (analysis by TLC),the mixture was allowed to reach room temperature and washed with 1MKOH. The layers were separated and the aqueous layer was furtherextracted with ethyl acetate (3×30 mL). The combined organic phases werewashed with brine and dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude solid was purified viacolumn chromatography with silica gel to obtain the appropriate2,3-diaminopyridine derivatives 9a or 9b.

N²-(3-Morpholin-4-yl-propyl)-pyridine-2,3-diamine (9a)

¹H NMR (400 MHz, CDCl₃) δ 1.84 (2H, quint, J=6.37 Hz), 2.48 (6H, q,J=6.48 Hz), 3.50 (2H, t, J=6.35 Hz), 3.73 (4H, t, J=4.64 Hz), 6.48 (1H,dd, J=5.11, 7.37 Hz), 6.81 (1H, dd, J=1.52, 7.37 Hz), 7.71 (1H, dd,J=1.52, 5.12 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 25.3, 41.72, 53.8, 58.2,67.0, 112.3, 121.2, 128.5, 138.8, 150.4.

2-Morpholin-4-yl-pyridin-3-ylamine (9b)

¹H NMR (400 MHz, CDCl₃) δ3.13 (4H, t, J=4.73 Hz), 3.86 (4H, t, J=4.58Hz), 6.85 (1H, dd, J=4.80, 7.73 Hz), 6.95 (1H, dd, J=1.65, 7.74 Hz),7.81 (1H, dd, J=1.64, 4.80 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 49.1, 67.4,119.7, 121.7, 135.4, 137.8, 150.7.

Synthesis of 9-ethyl-9H-carbazole-3-carboxylic acid (10)

In a 100 mL three neck round bottom flask equipped with a stirring barand reflux condenser, 9-ethyl-3-carbazolecarboxaldehyde (0.66 g, 3.0mmol), KMnO₄ (0.474 g, 3.0 mmol), and NaOH (0.120 g, 3.0 mmol) wereadded dissolved in water (10 mL). The reaction mixture was refluxed for3 h with vigorous magnetic stirring. As the reaction progresses, theintense purple color (manganese VII) will gradually disappear as it isreduced (to manganese IV) which precipitates as brown manganese dioxide.After 3 h, the reaction is allowed to reach room temperature, and themanganese dioxide precipitate was filtered with a Buchner funnel. Thefiltrate was pour into a separatory funnel and acidified with 1M HCl(until reach pH 6.5-7.5). The carbazole carboxylic acid precipitate 10was vacuum filtered and washed with water (2×50 mL). The product waspurified by recrystallization in CH₂Cl₂-MeOH (9:1). R_(f)=0.64. ¹H NMR(400 MHz, DMSO-d₆) δ 1.33 (3H, t, J=7.0 Hz), 4.48 (2H, q, J=6.74 Hz),7.26 (1H, t, J=7.35 Hz), 7.51 (1H, t, J=7.54 Hz), 7.65 (1H, d, J=3.74Hz), 7.68 (1H, d, J=4.26 Hz), 8.06 (1H, d, J=8.66 Hz), 8.27 (1H, d,J=7.94), 8.78 (1H, s), 12.52 (11H, bs); ¹³C NMR (100 MHz, DMSO-d₆) δ13.7, 37.2, 108.8, 109.5, 119.7, 120.7, 121.1, 121.9, 122.3, 122.5,140.2, 142.1, 168.0.

Synthesis of 9-Ethyl-9H-carbazole-3-carboxylic Acid[2-(3-morpholin-4-yl-propylamino)-pyridin-3-yl]-amide (11a)

In a 100 mL three-neck round-bottom flask was charged withN²-(3-Morpholin-4-yl-propyl)-pyridine-2,3-diamine derivative 9a (0.210g, 0.880 mmol), 9-ethyl-9H-carbazole-3-carboxylic acid 10 (0.239 g, 1.0mmol), HOBt (0.270 g, 2.0 mmol), and EDAC (0.3830 g, 2.0 mmol) dissolvedin THE (10 mL). The mixture was stirred for 10 min, then Et₃N (0.3 mL,2.0 mmol) was added. The mixture was stirred at room temperature for 16h. After the reaction was complete (analyzed by TLC), the solution waswashed with water (30 mL), and the product was extracted with CH₂Cl₂(3×20 mL). The combined organic phases were washed with brine and driedover anhydrous Na₂SO₄, filtered, and concentrated under reducedpressure. The crude was purified via column chromatography over silicagel and the product 11a was obtained as a white solid (0.180 g, 0.390mmol, 45%). TLC analysis in CH₂Cl₂-MeOH (9:1), R_(f)=0.52. ¹H NMR (400MHz, DMSO-d₆) δ 1.34 (3H, t, J=7.8 Hz), 1.72 (2H, t, J=6.8 Hz), 2.34(5H, s), 3.50 (4H, s), 4.50 (2H, q, J=7.1 Hz), 6.09 (1H, t, J=4.6 Hz),6.59 (1H, t, J=6.8 Hz), 7.28 (1H, t, J=7.6 Hz), 7.50 (1H, d, J=6.3 Hz),7.52 (1H, d, J=7.8 Hz), 7.69 (1H, d, J=8.9 Hz), 7.71 (1H, d, J=8.6 Hz),7.95 (1H, d, J=4.3 Hz), 8.14 (1H, d, J=8.1 Hz), 8.23 (1H, d, J=7.8 Hz)8.86 (1H, s); ¹³C NMR (100 MHz, DMSO-d6) δ 13.6, 25.8, 30.7, 37.2, 53.4,56.2, 66.1, 108.5, 109.6, 111.2, 119.3, 119.5, 120.5, 120.8, 121.7,122.4, 124.9, 125.8, 126.3, 133.6, 140.2, 141.4, 144.7, 154.0, 166.5.

Synthesis of 9-Ethyl-9H-carbazole-3-carboxylic Acid(2-morpholin-4-yl-pyridin-3-yl)-amide (11b)

In a 100 mL three-neck round-bottom flask was charged with2-Morpholin-4-yl-pyridin-3-ylamine derivative 9b (0.180 g, 1.0 mmol),9-ethyl-9H-carbazole-3-carboxylic acid 10 (0.1196 g, 0.5 mmol), HOBt(0.0675 g, 0.5 mmol), and EDAC (0.0958 g, 0.5 mmol) dissolved in THF (10mL). The mixture was stirred for 10 min, then Et₃N (0.3 mL, 2.0 mmol)was added. The mixture was stirred at room temperature for 16 h. Afterthe reaction was complete (analyzed by TLC), the solution was washedwith water (30 mL), and the product was extracted with CH₂Cl₂ (3×20 mL).The combined organic phases were washed with brine and dried overanhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Thecrude was purified via column chromatography over silica gel and theproduct 11b was obtained as a white solid (0.1271 g, 0.320 mmol, 32%).TLC analysis in CH₂Cl₂-MeOH (9:1), R_(f)=0.93. ¹H NMR (400 MHz, CDCl₃) δ1.49 (3H, t, J=7.1 Hz), 3.17 (4H, t, J=4.8 Hz), 3.99 (4H, t, J=4.8 Hz),4.44 (2H, q, J=6.8 Hz), 7.16 (1H, d, J=4.3 Hz), 7.17 (1H, d, J=4.44 Hz),7.36 (1H, t, J=7.6 Hz) 7.49 (1H, d, J=7.8 Hz), 7.53 (1H, d, J=8.3 Hz),7.55 (1H, d, J=8.1 Hz), 8.03 (1H, d, J=2.0 Hz), 8.05 (1H, d, J=1.8 Hz)8.14 (1H, d, J=1.8 Hz), 8.15 (1H, d, J=1.8 Hz), 8.71 (1H, d, J=1.8 Hz),8.85 (1H, d, J=1.8 Hz) 8.86 (1H, d, J=1.5 Hz), 9.06 (1H, s); ¹³C NMR(100 MHz, CDCl₃) δ 13.8, 37.9, 50.6, 67.6, 108.6, 109.0, 119.9, 120.6,120.9, 122.8, 123.1, 124.4, 124.8, 126.7, 127.3, 128.7, 140.7, 142.1,142.4, 152.7, 165.9.

Synthesis of orhto-diamide Derivatives 14a-h Synthesis of3-((9-ethyl-9H-carbazol-3-yl)carbamoyl)pyrazine-2-carboxylic AcidPrecursor (13) for Compounds 14a-d

In a 100 mL flask, 2,3-pyrazinedicarboxylic anhydride 12 (0.7505 g, 5mmol) was reacted with 2 (1.0511 g, 5 mmol) in dry THF (10 mL) stirredovernight at rt. The mixture was transferred to a separatory funnel andextracted with CH₂Cl₂ (3×10 mL) and NaOH (1N) (10 mL). The organic phasewas separated and HCl (1N) (10 mL) was added to the aqueous phase untila pH=2 was reached and a precipitate formed. The solid was filtered andwashed with water (20 mL), and concentrated under reduced pressure toprovide 13 (1.73 g, 4.8 mmol, 96%), as a yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 1.33 (3H, t, J=7.02 Hz), 2.51 (4H, d, J=1.59Hz), 4.46 (2H, q, J=7.01 Hz), 7.21 (1H, t, J=7.52 Hz), 7.47 (1H, t,J=7.66), 7.59 (1H, d, J=4.10 Hz), 7.61 (1H, d, J=3.48 Hz), 7.73 (1H, d,J=7.27 Hz), 8.58 (1H, s), 8.63 (2H, s); ¹³C NMR (100 MHz, DMSO-d₆) δ14.2, 37.5, 109.4, 109.7, 110.95, 119.1, 119.7, 120.6, 122.3, 122.6,126.2, 131.8, 136.8, 140.5, 141.8, 144.8, 145.8, 164.3.

Synthesis ofN²-(9-ethyl-9H-carbazol-3-yl)-N³-(2morpholinoethyl)pyrazine-2,3-dicarboxamide(14a)

In an oven dried 25 mL three-neck round bottom flask, 13 (0.0945 g, 0.20mmol) was dissolved in THF (8 mL). Then, 4-(2-aminoethyl)morpholine(28.9 μL, 0.22 mmol) was added with HOBt (0.02703 g, 0.20 mmol) and EDC(0.0460 g, 0.24 mmol). The mixture was stirred at room temperature for 3h. The mixture was transferred to a separatory funnel and extracted withCH₂C₂ (3×10 mL) and washed with NaHCO₃ (1N) (10 mL). The organic phasewas separated, dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure. The crude product was purified by silica gelcolumn chromatography with CH₂Cl₂-MeOH (via gradient starting of 99:1)to provide 14a (0.04 g, 0.085 mmol, 42.3%). R_(f)=0.52 ¹H NMR (400 MHz,CDCl₃) δ 1.42 (3H, t, J=7.2 Hz), 2.59 (3H, s), 2.74 (2H, t, J=5.6 Hz),3.69 (5H, m, J=4.0 Hz), 4.35 (2H, q, J=7.6 Hz), 7.22 (1H, t, J=7.6 Hz),7.26 (1H, s), 7.35 (1H, d, J=8.8 Hz), 7.38 (1H, d, J=8.0 Hz), 7.47 (1H,t, J=7.2 Hz), 7.68 (1H, d, J=8.4 Hz), 8.10 (1H, d, J=8.0 Hz), 8.56 (1H,s), 8.66 (1H, d, J=12.0 Hz), 9.52 (1H, s); ¹³C NMR (100 MHz, CDCl₃) δ36.0, 37.6, 53.2, 56.7, 66.5, 108.5, 108.5, 112.7, 118.8, 119.2, 120.7,122.8, 123.0, 125.9, 129.2, 137.4, 140.4, 143.4, 144.8, 145.1, 148.6,160.9, 165.7.

Synthesis ofN²-(9-ethyl-9H-carbazol-3-yl)-N³-(3-morpholinopropyl)pyrazine-2,3-dicarboxamide(14b)

In an oven dried 25 mL three-neck round bottom flask, 13 (0.0945 g, 0.20mmol) was dissolved in dried distilled THF (10 mL). Then,3-morpholinopropan-1-amine (43.80 μL, 0.30 mmol) was added with HOBt(0.02703 g, 0.20 mmol) and EDC (0.0460 g, 0.24 mmol). The mixture wasstirred at room temperature overnight. The reaction mixture wasconcentrated and no extraction to the crude was performed. The crudeproduct was purified by silica gel column chromatography withCH₂Cl₂-MeOH (via gradient starting of 9:1) to provide 14b (0.030 g,0.062 mmol, 31%). R_(f)=0.50. ¹H NMR (400 MHz)(CDCl₃) δ 1.40 (3H, t,J=7.19 Hz), 1.84 (2H, m), 2.47 (4H, s), 2.58 (2H, t, J=6.03 Hz), 3.59(4H, t, J=4.37 Hz), 3.69 (2H, q, J=5.67 Hz), 4.31 (2H, q, J=7.19 Hz),7.21, (1H, t, J=7.47 Hz), 7.26 (1H, s), 7.32 (1H, d, J=8.70 Hz), 7.37(1H, d, J=8.15 Hz), 7.47 (1H, t, J=7.31 Hz), 7.68 (1H, d, J=8.69 Hz),8.09 (1H, d, J=7.27 Hz), 8.44 (1H, d, J=4.78 Hz), 8.58 (1H, d, J=1.92Hz), 8.63 (1H, s), 9.45 (1H, s); ¹³CNMR (100 MHz, CDCl₃) δ 13.8, 24.4,37.5, 40.2, 53.6, 58.2, 66.9, 108.4, 108.5, 112.6, 118.8, 119.2, 120.8,122.8, 123.0, 125.8, 129.3, 137.3, 140.4, 143.5, 144.3, 145.7, 148.4,161.2, 165.1.

Synthesis of Pyrazine-2,3-dicarboxylic acid2-[(9-ethyl-9H-carbazol-3-yl)-amide]-3-[(3-imidazol-1-yl-propyl)-amide](14c)

In an oven dried 25 mL three-neck flask, 13 (0.0945 g, 0.20 mmol) wasdissolved in dried distilled THF (10 mL). Then,3-(1H-imidazol-1-yl)propan-1-amine (35.8 μL, 0.30 mmol) was added withHOBt (0.02703 g, 0.20 mmol) and EDC (0.0460 g, 0.24 mmol). The mixturewas stirred at room temperature overnight. No extraction was done. Thecrude product was purified by silica gel column chromatography withCH₂Cl₂/Methanol (via gradient starting of 9:1) to provide 14c (0.050 g,0.106 mmol, 53%). R_(f)=1.3. ¹H NMR (400 MHz, CDCl₃) δ 1.38 (3H, t,J=7.20 Hz), 2.06 (2H, m), 3.44 (2H, t, J=6.0 Hz), 4.08 (2H, t, J=7.20Hz), 4.27 (2H, q, J=7.2 Hz), 6.92 (2H, d, J=22.0 Hz), 7.19 (1H, t, J=7.6Hz), 7.30 (1H, d, J=8.0 Hz), 7.35 (1H, d, J=15.2 Hz), 7.46 (1H, t,J=7.20 Hz), 7.52, 7.99 (1H, d, J=11.2 Hz), 8.45 (2H, s), 8.50 (1H, s);9.72 (1H, s); ¹³C NMR (100 MHz, CDCl₃) δ 13.7, 31.0, 36.8, 37.5, 44.4,108.5, 108.5, 112.3, 118.8, 118.9, 120.6, 122.6, 122.9, 125.9, 128.9,129.3, 137.3, 140.3, 143.3, 144.6, 144.7, 148.3, 160.8, 165.9.

Synthesis ofN²-(9-ethyl-9H-carbazol-3-yl)-N³-(2-methoxyethyl)pyrazine-2,3-dicarboxamide(14d)

In an oven dried 25 mL three-neck round bottom flask, 13 (0.0945 g, 0.20mmol) was dissolved in dried distilled THF (10 mL). Then,3-(1H-imidazol-1-yl)propan-1-amine (19.13, 0.22 mmol) was added withEt₃N (69.74 μL, 0.50 mmol), HOBt (0.02703 g, 0.20 mmol) and EDC (0.0460g, 0.24 mmol). The mixture was stirred at room temperature overnight. Noextraction was performed. The crude product was concentrated andpurified by silica gel column chromatography with CH₂Cl₂-MeOH (viagradient starting of 9:1) to provide 14d (0.010 g, 0.024 mmol, 12.0%).R_(f)=0.54. ¹H NMR (400 MHz, CDCl₃) δ 1.44 (3H, t, J=7.2 Hz), 3.41 (3H,s), 3.67 (2H, t, J=5.60 Hz), 3.79 (2H, q, J=5.20 Hz), 4.38 (2H, q,J=7.20 Hz), 7.09 (11H, s), 7.22 (11H, t, J=12.8 Hz), 7.40 (2H, t, J=8.0Hz), 7.51J (1H, t, J=10.4 Hz), 8.11 (1H, d, J=7.6 Hz), 8.58 (1H, d,J=2.0 Hz), 8.70 (2H, q, J=2.4 Hz), 9.36J (1H, s); ¹³C NMR (100 MHz,CDCl₃) δ 13.0, 37.6, 39.7, 58.8, 70.9, 108.5, 112.8, 118.8, 119.2,120.82, 122.8, 123.1, 125.9, 129.3, 137.5, 140.5, 143.6, 144.6, 145.6,148.1, 161.0, 165.3.

Synthesis of 2-((9-ethyl-9H-carbazol-3-yl)carbamoyl)benzoic AcidPrecursor (13) for Compounds 14e-f

In a 100 mL one-neck flask, carbazole 2 (1.4812 g, 10 mmol), was reactedwith phthalic anhydride 12 (2.1021 g, 10 mmol) in dry THF stirred for 3h at room temperature. The reaction mixture was transferred to aseparatory funnel and extracted with CH₂C₁₂ (3×10 mL) and NaOH (1N) (10mL). The organic phase was separated, and HCl (1N) (10 mL) was added tothe aqueous phase until a pH=2 was reached and a precipitate formed. Thesolid was filtered and washed with water (30 mL), and concentrated underreduced pressure to provide 13 (2.78 g, 7.76 mmol, 77.6%), as a whitesolid. TLC showed the product to be pure: CH₂C₁₂-MeOH (9:1). R_(f)=0.45.¹H NMR (400 MHz, DMSO-d₆) δ 1.31 (3H, t, J=6.8 Hz), 4.51 (2H, q, J=8.4Hz), 7.19 (1H, t, J=8.0 Hz), 7.45 (3H, m), 7.56 (2H, t, J=4.0 Hz), 7.69(2H, d, J=8.4 Hz), 7.84 (1H, t, J=7.6 Hz), 8.10 (1H, d, J=3.2 Hz), 8.53(1H, s), 12.43 (1H, s); ¹³C NMR (400 MHz, DMSO-d₆) δ 13.7, 37.0, 109.0,109.2, 111.4, 118.6, 119.4, 120.2, 121.9, 122.2, 125.7, 128.7, 128.9,129.5, 129.6, 132.0, 134.9, 136.2, 138.1, 140.0, 166.3, 172.3.

Synthesis ofN¹-(9-ethyl-9H-carbazol-3-yl)-N²-(2-methoxyethyl)phthalamide (14e)

In a three-neck round bottom flask, 13 (0.0941 g, 0.20 mmol) wasdissolved in dry THE (6 mL). Then, 2-methoxyethanamine (18.11 μL, 0.30mmol) was added with HOBt (0.02703 g, 0.20 mmol) and EDC (0.0460 g, 0.24mmol). The mixture was stirred at room temperature overnight. Thereaction mixture was transferred to a separatory funnel and extractedwith CH₂Cl₂ (3×10 mL) and NaHCO₃ (1N) (10 mL). The organic phase wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure. The crude product was purified by silica gel columnchromatography with CH₂Cl₂-Methanol (via gradient starting of 99:1) toprovide 14e (0.0349 g, 0.084 mmol, 42%). TLC showed the product to bepure: CH₂Cl₂-MeOH (9:1). R_(f)=0.61. ¹H NMR (400 MHz, CDCl₃) δ 1.40 (3H,t, J=7.2 Hz), 3.24 (3H, s), 3.46 (2H, t, J=5.2 Hz), 3.61 (2H, q, J=5.2Hz), 4.33 (2H, q, J=7.2 Hz), 6.82 (1H, t, J=5.2 Hz), 7.21 (1H, t, J=7.6Hz), 7.32 (1H, d, J=8.4 Hz), 7.38 (1H, d, J=8.0 Hz), 7.45 (1H, d, J=7.2Hz), 7.50 (1H, t, J=3.6 Hz), 7.52 (1H, d, J=9.6 Hz) 7.65 (1H, d, J=8.8Hz), 7.85 (1H, d, J=9.2 Hz), 8.51 (1H, s), 9.30 (1H, s); ¹³C NMR (400MHz, CDCl₃) δ 13.8, 37.5, 40.0, 58.7, 60.4, 70.7, 108.4, 108.5, 112.7,118.69, 119.4, 120.7, 122.9, 123.0, 125.8, 127.8, 129.4, 130.2, 130.3,132.0, 134.6, 135.1, 137.2, 140.4, 166.3, 170.2.

Synthesis ofN¹-(9-ethyl-9H-carbazol-3-yl)-N²-(2-hydroxyethyl)phthalamide (14f)

In a three-neck round flask, 13 (0.0941 g, 0.20 mmol) was dissolved indry THE (5 mL). Then, 2-aminoethanol (19.13 μL, 0.22 mmol) was addedwith Et₃N (69.74 μL, 0.50 mL) followed by HOBt (0.0270 g, 0.20 mmol) andEDC (0.0460 g, 0.24 mmol). The mixture was stirred at room temperaturefor 2 hr. Then, DMF (23.1 μL, 0.30 mmol) was added and the reaction wasleft stirring for 1 hr. The reaction mixture was transferred to aseparatory funnel and extracted with EtOAc (3×10 mL) and NaHCO₃ (1N) (10mL). The organic phase was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude product was purified bysilica gel column chromatography with CH₂Cl₂-MeOH (via gradient startingof 99:1) to provide product 14f (50.0 mg, 0.125 mmol, 62.3%). TLC showedthe product to be pure: CH₂Cl₂-MeOH (9:1). R_(f)=0.50. ¹H NMR (400 MHz,CDCl₃) δ 1.38 (3H, t, J=7.2 Hz), 3.53 (2H, q, J=5.2 Hz), 3.73 (2H, t,J=4.8 Hz), 4.25 (2H, q, J=7.2 Hz), 7.11 (1H, t, J=5.2 Hz), 7.19 (1H, t,J=7.2 Hz), 7.26 (1H, d, J=8.8 Hz), 7.36 (1H, d, J=8.0 Hz), 7.40 (1H, d,J=6.8 Hz), 7.42 (1H, t, J=4.8 Hz), 7.47 (1H, t, J=7.2 Hz), 7.63 (1H, d,J=8.4 Hz), 7.68 (1H, d, J=8.0 Hz), 8.03 (1H, d, J=8.0 Hz), 8.43 (1H, s),9.27 (1H, s); ¹³C NMR (400 MHz, CDCl₃) δ 13.6, 37.1, 43.1, 60.7, 108.1,108.2, 112.6, 118.4, 119.5, 120.5, 122.5, 125.4, 127.3, 127.8, 129.4,129.7, 130.2, 134.9, 135.0, 136.9, 140.1, 167.4, 170.5.

Synthesis of2-((9-ethyl-9H-carbazol-3-yl)carbamoyl)cyclohexanecarboxylic AcidPrecursor (13) for Compounds 14g-h. F

In a 100 mL flask, carbazole 2 (1.2334 g, 8 mmol) was reacted with1,2-cyclohexanedicarboxylic anhydride (predominantly cis) 12 (1.2612 g,6 mmol) in dry THF stirred for 8 h at 70° C. The reaction mixture wasextracted with ethyl acetate (3×10 mL) and NaOH (1N). The organic phasewas separated and HCl (1N) (10 mL) was added to the aqueous phase untila pH=2 was reached and a precipitate formed. The solid was filtered andwashed with water (30 mL), and concentrated under reduced pressure toprovide 13 (1.06 g, 2.91 mmol, 48.5%), as a white solid. TLC showed theproduct to be pure: CH₂Cl₂-MeOH (9:1) R_(f)=0.45. ¹H NMR (400 MHz,CDCl₃) δ 1.29 (3H, t, J=7.2 Hz), 1.69 (2H, m), 2.12 (2H, m, J=8.8 Hz),2.50 (1H, s), 2.61 (1H, t, J=4.0 Hz), 2.95 (1H, q, J=4.4 Hz), 4.43 (2H,q, J=13.6 Hz), 7.16 (1H, t, J=7.6 Hz), 7.43 (1H, t, J=7.6 Hz), 7.52 (1H,t, J=9.2 Hz), 7.54 (1H, d, J=4.0 Hz), 7.56 (1H, d, J=3.6 Hz), 8.04 (1H,d, J=8.0 Hz), 8.43 (1H, s), 10.22 (1H, s); ¹³C NMR (400 MHz, CDCl₃) δ13.6, 21.5, 22.8, 24.1, 25.9, 27.87, 28.4, 38.9, 108.7, 109.0, 111.0,118.4, 119.0, 120.1, 121.8, 122.1, 125.6, 131.7, 135.9, 139.9, 172.4,175.7.

Synthesis of cyclohexane-1,2-dicarboxylic acid1-[(9-ethyl-9H-carbazol-3-yl)-amide]-2-[(2-morpholin-4-yl-ethyl)-amide(14g)

In a three-neck round bottom flask, 13 (0.0728 g, 0.20 mmol) wasdissolved in dried distilled THF (10 mL). Then, 2-morpholinoethanamine(44.6 μL, 0.34 mmol) was added with HOBt (0.02703 g, 0.20 mmol) and EDC(0.0460 g, 0.24 mmol). The mixture was stirred at room temperatureovernight. The reaction mixture was concentrated and no extraction wasdone. The crude product was purified by silica gel column chromatographywith CH₂Cl₂/Methanol (via gradient starting of 99:1) to provide 14g (60mg, 82.38%). TLC showed the product to be pure: CH₂Cl₂-MeOH (9:1).R_(f)=0.50. ¹H NMR (400 MHz, CDCl₃) δ 1.38 (3H, t, J=7.2 Hz), 1.50 (2H,m), 1.77 (2H, m), 1.96 (2H, q, J=3.6 Hz), 2.14 (1H, q, J=3.2 Hz), 2.28(4H, t, J=4.0 Hz), 2.37 (2H, t, J=2.8 Hz), 2.75 (1H, m), 3.02 (1H, q,J=4.4 Hz), 3.33 (2H, q, J=5.6 Hz), 3.59 (4H, t, J=1.6 Hz), 4.31 (2H, q,J=7.2 Hz), 7.20 (1H, t, J=7.6 Hz), 7.26 (1H, d, J=8.8 Hz), 7.37 (1H, d,J=8.0 Hz), 7.47 (1H, t, J=9.6 Hz), 7.49 (1H, d, J=8.8 Hz), 8.04 (1H, d,J=8.0 Hz), 8.36 (1H, s), 8.45 (1H, s); ¹³C NMR (100 MHz, CDCl₃) δ 13.8,23.0, 24.0, 24.5, 26.9, 27.8, 37.5, 39.4, 45.1, 45.5, 53.6, 57.7, 66.8,108.3, 108.45, 112.3, 118.6, 119.1, 120.6, 122.8, 122.9, 125.7, 130.2136.9, 140.3, 172.5, 174.8.

Synthesis of cyclohexane-1,2-dicarboxylic acid1-[(9-ethyl-9H-carbazol-3-yl)-amide]-2-[(3-morpholin-4-yl-propyl)-amide](14h)

In a three-neck round bottom flask, 13 (0.0728 g, 0.20 mmol) wasdissolved in dried distilled THF (10 mL). Then,3-morpholinopropan-1-amine (49.7 μL, 0.34 mmol) was added with HOBt(0.02703 g, 0.20 mmol) and EDC (0.0460 g, 0.24 mmol). The mixture wasstirred at room temperature overnight. The reaction mixture wasconcentrated and no extraction was performed. The crude product waspurified by silica gel column chromatography with CH₂Cl₂-MeOH (viagradient starting of 99:1) to provide 14h (0.06 g, 0.12 mmol, 61.1%).R_(f)=0.61. ¹H NMR (400 MHz, CDCl₃) δ 1.40 (3H, t, J=7.2 Hz), 1.56 (3H,m), 1.76 (1H, m), 1.89 (2H, m), 2.00 (2H, q, J=4.0 Hz), 2.09 (2H, q,J=3.2 Hz), 2.31 (6H, t, J=6.0 Hz), 2.71 (1H, m), 3.01 (1H, q, J=4.8 Hz),3.32 (2H, q, J=6.4 Hz), 3.62 (4H, t, J=4.8 Hz), 4.34 (2H, q, J=7.2 Hz),7.20 (1H, t, J=7.2 Hz), 7.30 (1H, d, J=4.4 Hz), 7.40 (1H, d, J=8.0 Hz),7.46 (1H, t, J=6.8 Hz), 7.52 (1H, d, J=8.8 Hz), 8.04 (1H, d, J=10.4 Hz),8.38 (1H, s), 8.43 (1H, s); ¹³C NMR (400 MHz, CDCl₃) δ 13.8, 23.0, 24.5,26.9, 27.8, 37.5, 39.4, 45.1, 45.5, 53.6, 57.7, 66.8, 108.3, 108.5,112.3, 118.6, 119.1, 120.6, 122.8, 122.9, 125.7, 130.2, 136.9, 140.3,172.5, 174.8.

General Remarks

All experiments were carried out in pre-dried glassware (≥1 h, 80-90°C.) under a nitrogen atmosphere. Nuclear magnetic resonance (NMR)spectra were obtained using a 400 MHz Bruker Avance UltraShield™spectrometer. ¹H (400 MHz) and ¹³C (100 MHz) NMR were recorded in CDCl₃or DMSO-d₆, unless otherwise used, and the chemical shift were expressedin ppm relative to CDCl₃ (7.26 for ¹H and δ 77.0 for ¹³C) or DMSO-d₆ (δ2.50 for ¹H and δ 39.5 for ¹³C) as the internal standard. ¹H NMR data isreported as position (δ), relative integral, multiplicity (s, singlet;d, doublet; t, triplet; q, quartet; dt, doublet of triplets; dd, doubletof doublets; dq, doublet of quartets; m, multiplet; br, broad peak),coupling constant (J, Hz), and the assignment of the atom. ¹³C NMR dataare reported as position (δ) and assignment of the atom. Microwavereactions were conducted in a CEM Discovery Microwave for DrugDiscovery, SP-1445.

Chemistry

Progress of the reaction was monitored via TLC analysis (General purposesilica gel on glass 5×20 cm with UV indicator, and visualized by UVfluorescent Spectroline E Series Ultraviolet lamps, in most casesfollowed by staining with I₂. The compounds were purified via columnchromatography over silica gel (70-230 mesh, 60 Å) with the appropriatesize column (24/40, 12 in.×0.5 in.) or (24/40, 12 in.×0.72 in.).

Synthesis ofN²-(5-(diethylamino)pentan-2-yl)-N⁴-(9-ethyl-9H-carbazol-3-yl)pyrimidine-2,4-diamine(32) via N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)with 2-Amino-5-diethylaminopentane

In a test tube, 30 (100 mg, 0.31 mmol) was dissolved in DMSO (1 mL).Cesium Carbonate (202 mg, 0.62 mol) and 2-Amino-5-diethylaminopentane(180.2 μL, 0.93 mol) were added. The mixture was stirred in a CEMDiscover Model Discover/Electromagnetic stir no. SP-1445 microwave at120° C. for 6 h. The reaction was transferred to a separatory funnel,and an extraction was done using CH₂C₁₂ (10 mL) and distilled H₂O (5mL). The aqueous phase and the organic phase were separated, and theaqueous phase was extracted with CH₂C₁₂ (5 mL). Afterwards, then theorganic phase was extracted with brine, dried over Na₂SO₄, filtered andconcentrated under vacuum on rotavap. The crude product was purified bysilica gel column chromatography (CH₂Cl₂/methanol 9:1 as eluent and100RL Et₃N) to obtain 32 in a yield of 0.03 g (0.08 mol, 21.8%). TLCanalysis showed the product to be pure: (CH₂Cl₂/methanol 3:1) Rf=0.5.

Synthesis ofN²-(3-(1H-imidazol-1-yl)propyl)-N⁴-(9-ethyl-9H-carbazol-3-yl)pyrimidine-2,4-diamine(33) with N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)and 1-(3-Aminopropyl)imidazole

In a test tube, 30 (100 mg, 0.31 mmol) was dissolved in DMSO (1 mL).1-(3-Aminopropyl)imidazole (37.0 μL, 0.31 mol) were added. The mixturewas stirred in the microwave at 160° C. for 3 h. The reaction wastransferred to a separatory funnel, and an extraction was done usingCH₂Cl₂ (10 mL) and distilled H₂O (5 mL). The aqueous phase and theorganic phase were separated, and the aqueous phase was extracted withCH₂Cl₂ (5 mL). Afterwards, the organic phase was extracted with brine,dried over Na₂SO₄, filtered and concentrated under vacuum on rotavap.The crude product was purified by silica gel column chromatography(CH₂Cl₂/methanol 9:1 as eluent and 100 μL Et₃N) to obtain 33 in a yieldof 41.7% (0.053 g, 0.13 mmol). TLC analysis showed the product to bepure: (CH₂Cl₂/methanol 3:1) Rf=0.53.

Synthesis ofN⁴-(9-ethyl-9H-carbazol-3-yl)-N²-(piperidin-2-ylmethyl)pyrimidine-2,4-diamine(34) with N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)and 2-(Aminomethyl)piperidine

In a test tube, 30 (100 mg, 0.31 mmol) was dissolved in 1,4-Dioxane (1mL). Cesium Carbonate (101 mg, 0.31 mol) and 2-(Aminomethyl)piperidine(37.6 μL, 0.31 mol) were added. The mixture was stirred in the microwaveat 160° C. for 3 h. The reaction was transferred to a separatory funnel,and an extraction was done using CH₂Cl₂ (10 mL) and distilled H₂O (5mL). The aqueous phase and the organic phase were separated, and theaqueous phase was extracted with CH₂Cl₂ (5 mL). Afterwards, the organicphase was extracted with brine, dried over Na₂SO₄, filtered andconcentrated under vacuum on rotavap. The crude product was purified bysilica gel column chromatography (CH₂Cl₂/methanol 9:1 as eluent and 100μL Et₃N) to obtain 34 in a yield of 16.1% (0.02 g, 0.05 mmol). TLCanalysis showed the product to be pure: (CH₂Cl₂/methanol 3:1) Rf=0.36.

Synthesis ofN⁴-(9-ethyl-9H-carbazol-3-yl)-N²-(2-(1-methylpyrrolidin-2-yl)ethyl)pyrimidine-2,4-diamine(35) with N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)and 2-(2-Aminoethyl)-1-methylpyrrolidine

In a test tube, 30 (100 mg, 0.31 mmol) was dissolved in DMSO (1 mL).Cesium Carbonate (202 mg, 0.62 mol) and2-(2-Aminoethyl)-1-methylpyrrolidine (74.0 L, 0.62 mol) were added. Themixture was stirred in the microwave at 120° C. for 6 h. The mixture wasstirred in the microwave at 120° C. for 6 h. The reaction wastransferred to a separatory funnel, and an extraction was done usingCH₂Cl₂ (10 mL) and distilled H₂O (5 mL). The aqueous phase and theorganic phase were separated, and the aqueous phase was extracted withCH₂Cl₂ (5 mL). Afterwards, the organic phase was extracted with brine,dried over Na₂SO₄, filtered and concentrated under vacuum on rotavap.The crude product was purified by silica gel column chromatography(CH₂Cl₂/methanol 9:1 as eluent and 100 μL Et₃N) to obtain 35 in a yieldof 0.04 g (0.10 mol, 31.1%). TLC analysis showed the product to be pure:(CH₂Cl₂//methanol 3:1) Rf=0.5

Synthesis of2-((4-((9-ethyl-9H-carbazol-3-yl)amino)pyrimidin-2-yl)amino) ethanol(36) with N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)and 2-aminoethanol

In a test tube, 30 (80 mg, 0.25 mmol) was dissolved in 1,4-Dioxane (3mL). DIPEA (87 μL, 0.5 mmol) and 2-aminoethanol (23 μL, 0.375 mmol) wereadded. The mixture was stirred in the microwave at 160° C. for 4-6 h.The reaction was transferred to a separatory funnel, and an extractionwas done using EtOAc (50 mL) and distilled H₂O (50 mL). The aqueousphase and the organic phase were separated, and the aqueous phase wasextracted with EtOAc (50 mL). Afterwards, the combined organic phase wasextracted with brine, dried over Na₂SO₄, and filtered. The flask was putin the rotovap to eliminate the dissolvent and concentrated undervacuum. The crude product was purified by silica gel columnchromatography (gradient 1%-10% methanol in dichloromethane) to obtain36 in a yield of 20.1 mg (0.056 mmol, 23.1%). TLC analysis showed theproduct to be pure: (CH₂Cl₂/MeOH 3:1).

Synthesis of2-(2-((4-((9-ethyl-9H-carbazol-3-yl)amino)pyrimidin-2-yl)amino)ethoxy)ethanol (37) withN-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30) and2-(2-aminoethoxy)ethanol

In a test tube, 30 (80 mg, 0.25 mmol) was dissolved in 1,4-Dioxane (3mL). DIPEA (87 μL, 0.5 mmol) and 2-(2-aminoethoxy)ethanol (38 μL, 0.375mmol) were added. The mixture was stirred in the microwave at 160° C.for 4-6 h. The reaction was transferred to a separatory funnel, and anextraction was done using EtOAc (50 mL) and distilled H₂O (50 mL). Theaqueous phase and the organic phase were separated, and the aqueousphase was extracted with EtOAc (50 mL). Afterwards, the organic phasewas extracted with brine, dried over Na₂SO₄, and filtered. The flask wasrotovap to eliminate the disolvent and concentrated under vacuum. Thecrude product was purified by silica gel column chromatography (gradient1%-10% methanol in dichloromethane) to obtain 38 in a yield of 49.9 mg(0.128 mmol, 50.1%). TLC analysis showed the product to be pure:(CH₂Cl₂/MeOH 3:1).

Synthesis ofN⁴-(9-ethyl-9H-carbazol-3-yl)-N²-(2-methoxyethyl)pyrimidine-2,4-diamine(38) with N-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30)and 1-methoxypropane

In a test tube, 30 (80 mg, 0.25 mmol) was dissolved in 1,4-Dioxane (3mL). DIPEA (87 μL, 0.5 mmol) and 2-methoxyethylamine (33 μL, 0.375 mmol)were added. The mixture was stirred in the microwave at 160° C. for 4-6h. The reaction was transferred to a separatory funnel, and anextraction was done using EtOAc (50 mL) and distilled H₂O (50 mL). Theaqueous phase and the organic phase were separated, and the aqueousphase was extracted with EtOAc (50 mL). Afterwards, the organic phasewas extracted with brine, dried over Na₂SO₄, and air filtered to thenrotovap and concentrated under vacuum. The crude product was purified bysilica gel column chromatography (gradient 1%-10% methanol indichloromethane) to obtain 38 in a yield of 53 mg (0.147 mmol, 58.9%).TLC analysis showed the product to be pure: (CH₂Cl₂/MeOH 3:1).

Synthesis of N²-(2,4-dimethoxybenzyl)-N⁴-(9-ethyl-9H-carbazol-3-yl)pyrimidine-2,4-diamine (39) withN-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30) and(2,4-dimethoxyphenyl)methanamine

In a test tube, 30 (80 mg, 0.25 mmol) was dissolved in 1,4-Dioxane(XmL). DIPEA (87 μL, 0.5 mmol) and 2,4-dimethoxybenzylamine (56 μL,0.375 mol) were added. The mixture was stirred in the microwave at 160°C. for 4-6 h. The reaction was transferred to a separatory funnel, andan extraction was done using EtOAc (50 mL) and distilled H₂O (50 mL).The aqueous phase and the organic phase were separated, and the aqueousphase was extracted with EtOAc (50 mL). Afterwards, the organic phasewas extracted with brine, dried over Na₂SO₄, and filtered. The flask wasrotovap to eliminate the dissolvent and concentrated under vacuum. Thecrude product was purified by silica gel column chromatography (gradient1%-10% methanol in dichloromethane) to obtain 39 in a yield of 28.5 mg(0.063 mmol, 25.2%). TLC analysis showed the product to be pure:(CH₂Cl₂/MeOH 3:1).

Synthesis of N⁴-(9-ethyl-9H-carbazol-3-yl)-N²-(2,4,6-trimethoxyphenyl)pyrimidine-2,4-diamine (40) withN-(2-chloropyrimidin-4-yl)-9-ethyl-9H-carbazol-3-amine (30) and2,4,6-trimethoxyaniline

In a test tube, 30 (80 mg, 0.25 mmol) was dissolved in 1,4-Dioxane (3mL). DIPEA (87 μL, 0.5 mmol) and 2,4,6-trimethoxyaniline (68 mg, 0.375mmol) were added. The mixture was stirred in the microwave at 160° C.for 4-6 h. The reaction was transferred to a separatory funnel, and anextraction was done using EtOAc (50 mL) and distilled H₂O (50 mL). Theaqueous phase and the organic phase were separated, and the aqueousphase was extracted with EtOAc (50 mL). Afterwards, the organic phasewas extracted with brine, dried over Na₂SO₄, and filtered. The flask wasrotovap to eliminate the dissolvent and concentrated under vacuum. Thecrude product was purified by silica gel column chromatography (gradient1%-10% methanol in dichloromethane) to obtain 40 in a yield of 71.4 mg(0.152 mmol, 60.9%). TLC analysis showed the product to be pure:(CH₂Cl₂/MeOH 3:1).

Synthesis of1-(5-chloro-2,4-dimethoxyphenyl)-3-(4-((9-ethyl-9H-carbazol-3-yl)amino)pyrimidin-2-yl)urea(41) with N⁴-(9-ethyl-9H-carbazol-3-yl)-6-methylpyrimidine-2,4-diamine(30a) and 1-chloro-5-cyanato-2,4-dimethoxybenzene

A 100 mL round flask was charged with 30a (96.22 mg) was dissolved inTHF (1 mL). 1-chloro-5-cyanato-2,4-dimethoxybenzene (66.22 mg, 0.31 mol)and Et₃N (127 μL, 0.93 mmol) were added. The mixture was stirredovernight at rt. The reaction was transferred to a separatory funnel,and an extraction was done using CH₂Cl₂ (10 mL) and distilled H₂O (5mL). The aqueous phase and the organic phase were separated, and theaqueous phase was extracted with CH₂Cl₂ (5 mL). Afterwards, the organicphase was extracted with brine, dried over Na₂SO₄, filtered andconcentrated under vacuum on rotavap. The crude product was purified bysilica gel column chromatography (CH₂Cl₂/methanol 9:1 as eluent and 100μL Et₃N) to obtain 41 in a yield of 18.7% (0.03 g, 0.06 mmol). TLCanalysis showed the product to be pure: (CH₂C₂/methanol 3:1) Rf=0.48.

Synthesis of(2-Chloro-6-methyl-pyrimidin-4-yl)-(9-ethyl-9H-carbazol-3-yl)-amine,HV-118

In a 250 mL three neck round bottom flask was charged with 1.23 g (7.55mmol) of 2,4-dichloro-6-methylpyrimidine 6 (previously purified withethyl acetate), 1.587 g (7.55 mmol) of 3-amino-9-ethylcarbazole 2, and1.05 mL (7.55 mmol) of triethylamine dissolved in THF. The mixture wasstirred for 1 h until all reagents dissolve. Then the solution wasrefluxed overnight at 76° C. After the reaction was complete (monitoredby TLC), water (100 mL) was added, and the product extracted with ethylacetate (3×40 mL). The combined organic phases were washed with brineand dried over anhydrous Na₂SO₄ (1 h), filtered, and concentrated underreduced pressure. The crude was purified via column chromatography oversilica gel, and the product HV-118 was obtained pure as a yellow solid(0.440 g, 1.31 mmol) in 17% yield. ¹H NMR (400 MHz, CDCl₃) δ 1.43 (3H,t, J=6.40 Hz), 2.40 (s, 3H), 4.36 (q, 2H, J=6.80 Hz), 6.61 (s, 1H), 7.20(d, 1H, J=1.1 Hz), 7.21 (d, 1H, J=5.93 Hz), 7.38 (d, 1H, J=9.23 Hz),7.39 (d, 1H, J=9.44 Hz), 7.46 (t, 1H, J=7.04 Hz), 7.60 (d, 1H, J=8.00Hz), 8.08 (d, 1H, J=7.80 Hz), 8.33 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ13.8, 23.9, 37.7, 100.1, 108.8, 109.3, 117.8, 119.2, 120.6, 122.3,123.6, 123.7, 126.4, 128.0, 138.5, 140.5, 160.2, 164.6, 168.5.

Synthesis ofN⁴-(9-Ethyl-9H-carbazol-3-yl)-6-methyl-N²-(3-morpholin-4-yl-propyl)-pyrimidine-2,4-diamine(8)

In a microwave test tube equipped with a magnetic stirrer, the compoundHV-118 (0.0508 g, 0.15 mmol) was dissolved in 2 mL of dioxane. Then,3-morpholinopropylamine 4 (0.023 mL, 0.16 mmol) and Cs₂CO₃ (0.1456 g,0.45 mmol) were added. The mixture was stirred for 1 h until allreagents dissolve completely. The mixture was heated to 160° C. for 2 h.After the reaction was complete (monitored by TLC), water was added (5mL), and the product extracted with ethyl acetate (4×10 mL). Thecombined organic phases were washed with brine and dried over anhydrousNa₂SO₄ (1 h), filtered, and concentrated under reduced pressure. Thecrude was purified via column chromatography over silica gel, and theproduct 8 was obtained pure as a pale yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 1.25 (br, 2H), 1.46 (t, 3H, J=7.20 Hz), 1.82 (quin, 2H, J=6.73Hz), 2.17 (d, 2H, J=4.44 Hz), 2.46 (t, 4H, J=7.08 Hz), 3.48 (q, 2H,J=6.61 Hz), 3.72 (t, 4H, J=4.62 Hz), 4.39 (q, 2H, J=7.20 Hz), 5.76 (s,1H), 6.87 (br, 1H), 7.23 (d, 1H, J=7.19 Hz), 7.25 (s, 1H), 7.35 (dd, 1H,J=1.90, 8.60 Hz), 7.39 (d, 1H, J=7.76 Hz), 7.42 (d, 1H, J=8.56 Hz), 7.49(t, 1H, J=8.06 Hz), 8.04 (s, 1H), 8.06 (d, 1H, J=7.59 Hz); ¹³C NMR (100MHz, CDCl₃) δ 13.8, 22.9, 26.3, 37.7, 39.9, 53.7, 56.7, 66.9, 92.7,108.7, 108.9, 116.7, 119.0, 120.5, 122.5, 123.0, 123.4, 126.1, 129.5,137.9, 140.5, 163.4.

The NMR data for various compounds according to the instant disclosurefollows.

Example 2: Synthesis of HV-107 (aka “Compound 11b”)

The instant example describes the synthesis of9-Ethyl-9H-carbazole-3-carboxylic acid(2-morpholin-4-yl-pyridin-3-yl)-amide (aka HV-107 or Compound 11b):

In a 100 mL three-neck round-bottom flask was charged with2-Morpholin-4-yl-pyridin-3-ylamine derivative 9b (0.180 g, 1.0 mmol),9-ethyl-9H-carbazole-3-carboxylic acid 10 (0.1196 g, 0.5 mmol), HOBt(0.0675 g, 0.5 mmol), and EDAC (0.0958 g, 0.5 mmol) dissolved in THF (10mL). The mixture was stirred for 10 min, then EtsN (0.3 mL, 2.0 mmol)was added. The mixture was stirred at room temperature for 16 h. Afterthe reaction was complete (analyzed by TLC), the solution was washedwith water (30 mL), and the product was extracted with CH₂Cl₂ (3×20 mL).The combined organic phases were washed with brine and dried overanhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Thecrude was purified via column chromatography over silica gel and theproduct 11B was obtained as a white solid (0.1271 g, 0.320 mmol, 32%).TLC analysis in CH₂Cl₂-MeOH (9:1), Rf=0.93. ¹HNMR (400 MHz, CDCl3) δ1.49 (3H, t, J=7.1 Hz), 3.17 (4H, t, J=4.8 Hz), 3.99 (4H, t, J=4.8 Hz),4.44 (2H, q, J=6.8 Hz), 7.16 (1H, d, J=4.3 Hz), 7.17 (1H, d, J=4.44 Hz),7.36 (1H, t, J=7.6 Hz) 7.49 (1H, d, J=7.8 Hz), 7.53 (1H, d, J=8.3 Hz),7.55 (1H, d, J=8.1 Hz), 8.03 (1H, d, J=2.0 Hz), 8.05 (1H, d, J=1.8 Hz)8.14 (1H, d, J=1.8 Hz), 8.15 (1H, d, J=1.8 Hz), 8.71 (1H, d, J=1.8 Hz),8.85 (1H, d, J=1.8 Hz) 8.86 (1H, d, J=1.5 Hz), 9.06 (1H, s); 13C NMR(100 MHz, CDCl3) δ 13.8, 37.9, 50.6, 67.6, 108.6, 109.0, 119.9, 120.6,120.9, 122.8, 123.1, 124.4, 124.8, 126.7, 127.3, 128.7, 140.7, 142.1,142.4, 152.7, 165.9.

Example 3: Synthesis of HV-118

The instant example describes the synthesis of(2-Chloro-6-methyl-pyrimidin-4-yl)-(9-ethyl-9H-carbazol-3-yl)-amine (akaHV-118):

In a 250 mL three neck round bottom flask was charged with 1.23 g (7.55mmol) of 2,4-dichloro-6-methylpyrimidine 6 (previously purified withethyl acetate), 1.587 g (7.55 mmol) of 3-amino-9-ethylcarbazole 2, and1.05 mL (7.55 mmol) of triethylamine dissolved in THF. The mixture wasstirred for 1 h until all reagents dissolve. Then the solution wasrefluxed overnight at 76° C. After the reaction was complete (monitoredby TLC), water (100 mL) was added, and the product extracted with ethylacetate (3×40 mL). The combined organic phases were washed with brineand dried over anhydrous Na₂SO₄ (1 h), filtered, and concentrated underreduced pressure. The crude was purified via column chromatography oversilica gel, and the product HV-118 was obtained pure as a yellow solid(0.440 g, 1.31 mmol) in 17% yield. ¹H NMR (400 MHz, CDCl₃) δ 1.43 (3H,t, J=6.40 Hz), 2.40 (s, 3H), 4.36 (q, 2H, J=6.80 Hz), 6.61 (s, 1H), 7.20(d, 1H, J=1.1 Hz), 7.21 (d, 1H, J=5.93 Hz), 7.38 (d, 1H, J=9.23 Hz),7.39 (d, 1H, J=9.44 Hz), 7.46 (t, 1H, J=7.04 Hz), 7.60 (d, 1H, J=8.00Hz), 8.08 (d, 1H, J=7.80 Hz), 8.33 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ13.8, 23.9, 37.7, 100.1, 108.8, 109.3, 117.8, 119.2, 120.6, 122.3,123.6, 123.7, 126.4, 128.0, 138.5, 140.5, 160.2, 164.6, 168.5.

Example 4: Scheme 1 Compounds

The growth inhibitory potency against MCF-7 and MDA-Mb-231 breast cancercells was tested and the resulting data is on Table 1. Among the twoseries of compounds 3a-d and 4a-f, compounds 4a and 4c-e demonstratemoderate antiproliferative activity with GI50 in the range of 13.4-28.3pM on MCF-7 cancer cell line. As for MDA-MB-231 cell line, compounds 3dand 4d-e showed GI50 in the range of 18-19.3 μM, and the remainingcompounds showed GI above 50 pM. The three-carbon atom aliphatic chainconnected between the morpholine group and the pyridine ring, as seen incompound 3d, showed a moderate increase in growth inhibitory potencyagainst MDA-MB-231 cell line. The effect of substituting the aminocarbazole at the 2-position of the pyridine ring and amide-linked withprimary aliphatic amines at 3-position, as seen for compounds 4a-e,resulted in improved cytotoxicity against both cancer cell lines.

TABLE 1 Growth inhibition and anti-migration activity for compounds 3a-dand 4a-f on MCF-7 and MDA-MB-231 cell lines. GI₅₀ (μM)^(a) MDA-Migration Cmpd R = MCF-7 MB-231 (%)^(b,c) 3a

>50 >50  93 ± 11.67 3b

>50 >50  68 ± 10.54 3c

>50 >50 99 ± 1.89 3d

>50   19 76 ± 7.87 MBQ-5

>50 >50 54 ± 8.75 4a

  28 >50 99 ± 1.28 4b

>50 >50 95 ± 9.80 4c

  27 >50 93 ± 2.01 4d

  13   18  91 ± 14.10 4e

  14   18 99 ± 4.12 4f

>50 >50 92 ± 8.55 EHop-016   14   15 67 ± 7.55 ^(a)GI₅₀ = compoundconcentration required to inhibit MDA-MB-231 proliferation by 50% after24 h treatment. Values are expressed as the mean of triplicateexperiments, and standard deviation (SD) are <10%. ^(b)After 24 h,MDA-MB-231 cellular migration was determined by measuring the distancetraveled from the edge of the scratch toward the center of the scratch,relative to control. ^(c)Percent relative migration values at 10 μM arethe average of three independent experiments.

Example 5: Scheme 2 Compounds

To test the effect of reversing the orientation of the amide linkage atposition-3 of the pyridine ring, a series 2,3-diamino-substitutedpyridine derivatives represented by 7a-c and 11a-b was synthesized(scheme 2). Among the five compounds synthesized, three compounds (7a-band 11a) exhibited moderate anti-proliferative activity, and forcompounds (7c) and (11b) minimal cytotoxicity was observed atconcentrations ≤50 μM in MDA-MB-231 cancer cells (see Table 2).

TABLE 2 Growth inhibition and anti-migration activity for compounds 7a-cand 11a-b on MCF-7 and MDA-MB-231 cell lines. GI₅₀ (μM)^(a) MDA-Migration Cmpd R = MCF-7 MB-231 (%)^(b,c)  7a

  15   23 99 ± 4.29  7b

  41   31  81 ± 11.12  7c

  23 >50 81 ± 8.27 11a

  32   39  89 ± 10.10 11b (aka HV-107)

>50 >50 66 ± 7.43 ^(a)GI₅₀ = compound concentration required to inhibitMDA-MB-231 proliferation by 50% after 24 h treatment. Values areexpressed as the mean of triplicate experiments, and standard deviation(SD) are <10%. ^(b)After 24 h, MDA-MB-231 cellular migration wasdetermined by measuring the distance traveled from the edge of thescratch toward the center of the scratch, relative to control.^(c)Percent relative migration values at 10 μM are the average of threeindependent experiments.

The effect of reversing the amide linkage, as with 3d compared withcompound 11a, showed no improvement in the growth inhibition potencyagainst MDA-MB-231 cancer cells. Additionally, the effect of replacingthe aliphatic amide group at the 3-position with an aromatic ring, wasalso explored by synthesizing compound 7b. This compound exhibitedmoderate antiproliferative effect similar to 11a. Within the reverseseries, the high anti-migratory activity of compound 11b is comparableto that of 3b, and with the parent compound EHop-016. Addition of athree-carbon atom aliphatic chain linker between the pyridine ring (atthe 2-position) and the morpholine group, as for 3d and 11a, results insome loss of anti-migratory activity. Moreover, the effect of 11b wastested on Rac activity, using an ELISA-based Rac activity assay andpulldowns assay. Results show that at 250 nM, 11b inhibits Racactivation by 55% in MDA-MB-231 and MDA-MB-435 cancer cells.

Example 6: Scheme 3 Compounds

By replacing the central pyrimidine core with ortho-diamide buildingblock, as represented in the general synthetic scheme 3, the compoundsare forced to adopt a U-shaped conformation, which was hypothesized tolead to enhanced binding to Rac1. Preliminary dockings demonstratedindeed lower binding energies for this class of compounds, and as thisimplied an increased potency, this was a good reason to synthesize andtest a series of ortho-diamide compounds containing the carbazole group,that was strongly correlated with Rac1 inhibitory activity. Utilizingthree different anhydride building blocks, the ortho-amidocarboxylicacids 14a-h were synthesized as represented in scheme 3 via a simplenucleophilic ring-opening reaction of the cyclic anhydrides 12 with3-amino-9-ethylcarbazole 2. After acidic work-up, extraction and removalof the solvent, the desired products 13 could be obtained essentiallypure in yields of 48 to 78%. Each of the ortho-amide carboxylic acids 13was reacted with a variety of amines in order to obtain theortho-diamide products 14a-h in yields of 43 to 62%.

An in vitro analysis demonstrated that the ortho-diamide derivatives didnot show growth inhibition in MCF-7 and MDA-MB-231 cell lines (see Table3). Hence, these specific derivatives are not viable anti-cancertherapeutic agents. Furthermore, the ortho-diamide derivatives werestudied for anti-migratory effects using the wound-healing assay inMDA-MB-231 cell line. After 24 h of treatment at 10 μM, no migrationinhibition could be seen. In general, these SAR results furtherdemonstrate that for anti-migratory potency in this series of compounds,a morpholine-like substituent is preferred at position-2 of the pyridinering with the carbazole group linked with an amide at position-3.

TABLE 3 Growth inhibition and anti-migration activity for compounds14a-h on MCF-7 and MDA-MB-231 cell lines.               Cmpd              R =

             GI₅₀ (μM)^(a)          MDA-  MCF-7  MB-231             Migration (%)^(b,c) 14a

>50 >50 91 ± 5.23 14b

>50 >50 94 ± 5.87 14c

>50 >50 99 ± 4.50 14d

>50 >50 100 ± 5.20  14e

>50 >50 99 ± 2.57 14f

>50 >50 98 ± 1.83 14g

>50 >50 99 ± 1.63 14h

>50 >50 100 ± 0.56  ^(a)GI50 = compound concentration required toinhibit MDA-MB-231 proliferation by 50% after 24 h treatment. Values areexpressed as the mean of triplicate experiments, and standard deviation(SD) are <10%. ^(b)After 24 h, MDA-MB-231 cellular migration wasdetermined by measuring the distance traveled from the edge of thescratch toward the center of the scratch, relative to control.^(c)Percent relative migration values at 10 μM are the average of threeindependent experiments.

Example 7: Sulforhodamine B (SRB) Assay

Various compounds were tested to evaluate the dose response curve foreffect on MCF-7 cancer cells and MDA-MB-231 cancer cells. MCF-7 andMDA-MB-231 cells were cultured in 10% Minimum Essential Medium Eagle(MEME) supplemented with Earle's Balanced Salt Solution (EBSS),Non-essential Amino acids (NEAA), Sodium pyruvate, Pen/Strep,L-glutamine and Fetal Bovine Serum (FBS) at 37° C. in 5% CO₂.

Sulforhodamine B (SRB) Assay: A stock solution of compounds was preparedat 50 mM in 100% DMSO. For cells preparation, a flask of 75 cm² or 25cm² were used for 2.6×10⁵ cells or 1.44×10⁵ cells respectively with an80-90% of confluence. Cells were washed with PBS and trypsinized. Theconcentration of cells was determined using a 1:2 dilutions with TrypanBlue and a hemocytometer. After cell count, the concentration wasadjusted to have a 1.9×10⁴ cells/well. Approximately 100 μM of cellssuspension, compounds, control positive and control negative were addedin triplicates to a 96 well plate. Positive controls used wereellipticine, doxorubicin, and/or vincristine, and the negative controlwas DMSO 0.1%. All compounds at 50, 25, 12.5, 6.3, and 1.6 μM wereincubated with cells at 37° C. for 24 hrs. For fixation, cold TCA 50%was used and incubated at 4° C. for 1 hr. Wells were washed and driedprior to tincture with 100 μL of SRB 0.4%. To remove excess of SRB,acetic acid was used. For analysis, TRIS-BASE Solution (pH=10.5) wasused and shacked prior to reading using an ELISA reader at 510 nm andthe software SoftMax Pro 4.8.

For each compound, 50% growth inhibition (GI₅₀) was calculated fromSigmoidal dose-response curves (variable-slope) that were generated withdata obtained from experiments carried out in triplicates (GI₅₀ valueswere generated with GraphPad Prism V. 6.02, GraphPad Software, Inc.).The results are presented in FIGS. 4A-4B (MCF-7 cancer cells) and FIGS.5A-5B (MDA-MB-231 cancer cells).

Example 8: Wound Healing Assay (Scratch Method)

The experimental data of anti-migratory activities for compounds 3a-dand 4a-f were obtained using MDA-MB-231 breast cancer cells. Results aresummarized in Table 1 above. For the migration assay, a wound-healingassay (scratch-wound assay) was utilized in the presence of vehicle(0.02% DMSO) or compounds at 10 pM. FIG. 6 shows the results of themigration assay.

Among the eleven compounds synthesized and examined, two compounds 3band 3d exhibited 32% and 24% antimigratory activity, respectively.However, compounds 3a, 3c, and 4a-f inhibited migration in the range of1-9%. Using the same experimental conditions to determine theanti-migratory potential, the parent compound EHop-016 exhibitedanti-migratory activity of 33% at 10 pM and 17% at 5 pM. Despite thefact that compound 3b showed minor anti-proliferative activity againstMDA-MB-231 at <50 pM, the compound exhibited comparable anti-migratoryactivity with the parent EHop-016. However, EHop-016 has beendemonstrated that at concentrations >10 pM reduce cell viability inMCF10A mammary epithelial cell lines.

Prior to assays, cells were grown until 80-90% confluence was observed.A 75 cm2 flask was used for 2.6×105 cells/mL in 10 mL and for a 25 cm2flask 1.44×105 cells/mL in 5 mL. The cells were washed with PBS toremove all traces of FBS. 2 mL was added for a 25 cm2 flasks or 4 mL fora 75 cm2 flasks trypsin, and incubated 5-10 min at 37° C. At the end ofthe incubation time, cells were re-suspended and counted withhemocytometer using 1:2 dilutions with Trypan blue. Subsequently, cellviability was calculated. In a 12 multiwell plate, the plastic patternwas used to draw a fine space with a fine marker on the bottom of theplate. Cells were seeded at 1.5-2.2×10⁵ cells/mL in 1 mL and incubatedfor 24 h. Cells were then rinsed with PBS and incubated in starvingmedia (0.5% FBS) overnight. All controls and drugs were tested intriplicate. The negative control for each drug was prepared according tothe drug's DMSO concentration.

Drugs were diluted and the final concentration at each well was 10 pM.The wound was made using a sterile pipette tip of 200 pL. Cells werethen rinsed very gently with media without FBS and media with negativecontrols was added. After a 24 h incubation, the gap distance wasevaluated using the software Lumera Infinity Analyze 6.4.0. Pictureswere taken at 0, 8, 12 and 24 h using a 10× objective. The percentage ofmigration was calculated using the following formula:100−[(X ₀ /{umlaut over (X)} ₀)]*100 for time 0 h measurements100−[(X ₂₄ /{umlaut over (X)} ₀)]*100 for time 24 h measurements

The experimental data of anti-migratory activities for compounds 3a-dand 4a-f were obtained using MDA-MB-231 breast cancer cells, and resultsare summarized in Table 1. For the migration assay, we used awound-healing assay (scratch-wound assay)²² in the presence of vehicle(0.02% DMSO) or compounds at 10 μM (FIG. 6). Among the ten compoundssynthesized and examined, two compounds 3b and 3d exhibited 32% and 24%anti-migratory activity, respectively (Table 1). However, compounds 3a,3c, and 4a-f inhibited migration in the range of 1-9%. Using the sameexperimental conditions to determine the anti-migratory potential, theparent compound EHop-016 exhibited anti-migratory activity of 33% at 10μM and 17% at 5 μM. Despite the fact that compound 3b showed noanti-proliferative activity against MDA-MB-231 at ≤50 μM, the compoundexhibited comparable anti-migratory activity with the parent EHop-016.However, EHop-016 has been demonstrated that at concentrations ≥10 μMreduce cell viability in MCF10A mammary epithelial cell lines.

Example 9: Molecular Docking Assay

All compounds were drawn using ChemDraw Ultra 7.0 and energy-minimizedwith MOP AC AMI inChem3D Ultra 7.0. All fdes were saved as pdb fileformat. For molecular docking, Autodock 4.0 with Autodock Tools 1.5.4 asa graphical interface was used. The PDB files of the Rac1 protein wereobtained from the Protein Data Bank (PDB). For instance, the Rac1-Tiamcomplex (code: foe pdb) X-ray structure was used for the study. The Tiamstructure, water, metals, and any other molecule that is not part of theprotein were removed. The network of amino acids inside or surroundingTrp56 of Rac1 were set to be rigid. The grid box coordinates werelocated at the center of Trp56. The AutoDock Tools program was used toprepare the GEF-interacting region of Rac1 and the compounds for dockingusing 40×40×40 Å with a grid spacing of 0.375 Å, and a flexible dockingof 50 GA runs. The population size was set to 150 and the maximum numberof energy evaluation was set to 2,500,000.

The EHop-016 is a flexible molecule that can adopt differentconformations and interact with Rac1 in multiple conformations withinthe GEF-RacI site. Nevertheless, in docking studies the most favorableconformation that adopts EHop-016 in Rac1 is in a “U-bent” conformation.Docking of compound 11b with Rac1 shows the morpholine group and thepyridine ring in close contact with residues Val36 and Asp38. Moreover,the carbazole moiety points toward the hydrophobic pocket created byVal36 and Ala59. Docking of compound 3b positions the morpholine groupdeep into the pocket of Rac binding site and in close contact to Trp56.The pyridine ring forms hydrogen bonding with the peptide backbone ofAsp38 and Asn39.

Example 10: Effect of HV-107 on Cell Viability

HV-107 significantly inhibited the viability of MDA-MB-231 andMDA-MB-435 cells at ≥500 nM, while showing minimal toxicity towardsMCF10A cells. Cells were treated with ehicle control (0.5% DMSO) orvarying concentrations of HV-107 (250-8000 nM) for 24 or 48 h. Cellviability was measured using the CellTiter 96® AQueous One Solution CellProliferation Assay from Promega, as per manufacturer's instruction.Relative cell viability is presented for non-cancerous mammaryepithelial cells MCF10A (FIG. 7) or metastatic breast cancer cellsMDA-MB-231 or MDA-MB-435. N=3, each with three technical replicates;Error bars represent ±SEM; * p≤0.05.

Example 11: Effect of HV-107 on Rac Activation

The effects of HV-107 on the inhibition of Rac activation were tested bypulldown assays.

In FIG. 8A and FIG. 8B, human breast cancer cells MDA-MB-231 andMDA-MB-435, respectively, were treated for 24 h with 0, 250, or 500 nMHV-107. After treatment, total protein was extracted and equal amountsof proteins subjected to pulldown assays using the p21-binding domain ofPAK to isolate the GTP bound (active) Rac. Samples were then westernblotted for total and active Rac and positive bands quantified usingimage J. N=2; error bars represent ±SEM; * p≤0.05.

Results show that at 250 nM, HV-107 inhibits Rac activation by 60%.Therefore, HV-107 significantly inhibits Rac activation in metastaticbreast cancer cells with approximately 4× higher efficiency whencompared to the lead compound Ehop-016.

Example 12: Effect of HV-118 on Cell Viability

Similarly, HV-118 significantly inhibited the viability of MDA-MB-231and MDA-MB-435 cells at ≥50 nM, and had minimal effects on MFC10A cellviability. Cells were treated with vehicle control (0.5% DMSO) orvarying concentrations of HV-118 (10-500 nM) for 48 h. Cell viabilitywas measured using the CellTiter 96® AQueous One Solution CellProliferation Assay from Promega, as per manufacturer's instruction.Relative cell viability is presented for human non-cancerous mammaryepithelial cells MCF10A (FIG. 9) or human metastatic breast cancer cellsMDA-MB-231 or MDA-MB-435. N=3, each with three technical replicates;error bars represent ±SEM; * p≤0.05.

Example 13: Effect of HV-118 on Rac Activation

The effects of HV-118 on the inhibition of Rac activation were tested bypulldown assays.

In FIG. 10A and FIG. 10B, human breast cancer cells MDA-MB-231 andMDA-MB-435, respectively, were treated for 24 h with 0, 25, 50, or 100nM HV-118. After treatment, total protein was extracted and equalamounts of proteins subjected to pulldown assays using the p21-bindingdomain of PAK to isolate the GTP bound (active) Rac. Samples were thenwestern blotted for total and active Rac and positive bands quantifiedusing image J. N=3-6; error bars represent ±SEM; * p≤0.05.

Results show that at HV-118 has a similar effect at 100 nM in MDA-MB-231and MDA-MB-435 cells. Therefore, HV-118 significantly inhibits Racactivation in metastatic breast cancer cells with approximately 10×higher efficiency when compared to the lead compound Ehop-016.

Example 14: Synthesis of EHop-016 Derivatives with Modifications inBuilding Block C

For the synthesis of EHop-016 derivatives with modifications in buildingblock C, an optimized procedure for the synthesis of EHop-016 wasutilized (see U.S. Pat. No. 8,884,006, herein incorporated in itsentirety). FIG. 11A describes Scheme 1B, a two-step synthetic procedurefor the synthesis of EHop-016 and derivatives. The two-step syntheticscheme starts in “step a” with the synthesis of EHop-014 (3) fromdichloropyrimidine (1) and 3-amino-9-ethyl-carbazole (2). In theoptimized procedure, first dichloropyrimidine as obtained from thesupplier, is purified by slurrying in ethyl acetate for one hour.Insoluble solids (hydrolyzed impurities) are filtered and the ethylacetate is removed via rotary evaporation. Subsequently, purified 1 isreacted with carbazole 2 via reflux in tetrahydrofuran in the presenceof 1.2 equivalents triethyl amine. Compared with the previous method,workup was simplified by addition of water and ethyl acetate, which ledto the precipitation of 3. Filtration over a Buchner funnel providedcompound 3 without column chromatography as essentially the single4-substituted regioisomer in a yield of 52.1%. Reaction of product 3with different amines representing building block C, in the presence ofeither cesium carbonate or triethylamine amine in dioxane or dimethylsulfoxide under microwave heating at 120 to 160° C. (step b) providedthe desired EHop-016 derivatives. A summary of the products synthesized,together with their yields is provided in Table 4.

TABLE 4 Derivatives of EHop-016 with modifications in building block Csynthesized via Scheme 1B and Scheme 2B. Reaction Yield Entry# Buildingblock C conditions Product (%)  1

1,4-Dioxane Cs₂CO₃ Reflux 120 to 160° C.

  EHop-016 52.1  2

DMSO Cs₂CO₃ MW 120° C. for 6 h

  32 21.8  3

DMSO Cs₂CO₃ MW 160° C. for 3 h

  33 41.7  4

1.4-Dioxane Cs₂CO₃ MW 160° C. for 3 h

  34 16.1  5

DMSO Cs₂CO₃ MW 120° C. for 6 h

  35 31.1  6

1,4-Dioxane DIPEA MW 160° C. 4-6 hrs

  36 23.1  7

1,4-Dioxane DIPEA MW 160° C. 4-6 hrs

  37 50.1  8

1,4-Dioxane DIPEA MW 160° C. 4-6 hrs

  38 58.9  9

1,4-Dioxane DIPEA MW 160° C. 4-6 hrs

  39 25.2 10

1,4-Dioxane DIPEA MW 160° C. 4-6 hrs

  40 60.9 11

Et₃N THF Stir, rt overnight

  41 18.7

An additional example of a compounds with modification in building blockC was synthesized via a different procedure as represented in Scheme 2B(see FIG. 11B). The compounds that were synthesized were tested fortheir biological activity by determination of their cytotoxicity inMDA-MB-231 breast cancer cell lines as shown in Table 5. Thecytotoxicity's are expressed as IC₅₀, which indicates the concentrationat which 50% of the cell growth is inhibited. Furthermore, theinhibition of cell migration was tested at a concentration of 0.2×IC₅₀,or, when the IC₅₀ was >50 μM at a concentration of 10 μM. Forcomparison, Table 5 also includes the Lipinski and drug-likenessparameters as calculated via the MolSoft platform.

TABLE 5 Biological activity and physicochemical parameters of EHop-016and compounds and 2-11. Entry IC₅₀ Migration MW Number Number Drug #Compound Structure (μM) (%) (g/mol) MolLogP of HBA of HBD likeness  1

  EHop-016   15.74  63.36 (5 μM)  95.36 (1 μM) 430.55 4.62 4 2   0.51  2

  32    9.00  99.87 (1.8 μM) 444.30 6.60 3 2   0.60  3

  33    7.57 100.12 (1.51 μM) 411.22 4.35 3 2 −0.25  4

  34    1.97 100.45 (0.39 μM) 400.24 4.74 3 3   0.47  5

  35   19.13  89.13 (3.8 μM) 414.25 5.36 3 2   0.37  6

  36   14.61  94.85 (2.92 μM) 347.41 3.74 3 3 −0.39  7

  37 >50   100.39 (3.07 μM) 391.47 3.56 4 3 −0.23  8

  38 >50    96.74 (2.8 μM) 453.51 6.13 4 2   0.02  9

  39 >50   102.95 (0.92 μM) 361.44 4.32 3 2 −0.25 10

  40   7.7  80.15 (1.54 μM) 469.53 6.10 5 2   0.06 11

  41   10.9   77.34 (1 μM) 516.98 6.35 5 3   0.24

Example 15: Migration Assays for Compounds 5, 10, and 11 Compared toEHop-016

Migration assays were performed via the Scratch Method as describedpreviously. To illustrate a typical result, FIG. 12A shows photographsobtained with EHop-016 at concentrations of 1.0 and 5.0 μM, whichdemonstrates inhibition of migration of 5% and 37%, respectively.

Compounds with modifications in Building Block C demonstratinginhibition of migration at a concentration of 0.2×IC₅₀ includedcompounds 5, 10 and 11, as shown in FIG. 12B.

Example 16: Synthesis of HV-118 and EHop-016 Derivative 8

To explore new EHop-016 derivatives with minor modifications at thepyrimidine core building block A, compound HV-118 was synthesized (seeScheme 3B; FIG. 13). This compound was synthesized following essentiallythe same procedure as depicted in scheme 1 for the synthesis ofEHop-016. However, instead of using precursor 2,4-dichloropyrimidine 1,a commercially available 2,4-dichloro-6-methylpyrimidine 6 was used.

The pyrimidine 6 is reacted with carbazole 2 via reflux intetrahydrofuran in the presence of 1.2 equivalents triethylamine.Compared with previous purification procedures, to obtain 7 (HV-118) thecrude was purified using silica gel chromatography (5:1 to 1:1,hexane-ethyl acetate), and the pure compound was collected in 17% yield.Reaction of product 7 with amine 4, representing building block C, inthe presence of cesium carbonate in dioxane under microwave heating at120 to 160° C. provided the desired EHop-016 derivative 8. Byintroducing a methyl group in position-6 of the pyrimidine, hydrophobicinteractions with aromatic residues at the binding site of Rac may beimproved. Consequently, such derivatives are hypothesized to be morepotent Rac inhibitors and better anti-migratory compounds.

Example 17: Biological Effect of Compound 8

Compound 8 exhibited comparable growth inhibition potency to EHop-016,with GI₅₀=9.5 μM in MCF-7 cancer cell line and GI₅₀=6 μM in SH-SY5Yneuroblastoma cells (see FIG. 14A and FIG. 14B, respectively).

Example 18: Characteristics of EHOP-016 Derivatives

A panel of novel Ehop-016 derivatives was tested for their efficacy toinhibit Rac activation and selectively inhibit metastatic breast cancercell viability, while presenting minor toxicity to non-cancerous mammaryepithelial cells (MCF10A). Two Ehop-016 derivatives, HV-107 and HV-118,that are toxic to MDA-MB-231 and MDA-MB-435 metastatic breast cancercells, show minimal or no inhibition of non-cancerous mammary epithelialcell viability at the concentrations tested. The effects of HV-107 andHV-118 on the inhibition of Rac activation were tested by pulldownassays. Results show that at 250 nM, HV-107 inhibits Rac activation by60%; whereas HV-118 has a similar effect at 100 nM in MDA-MB-231 andMDA-MB-435 cells.

Subsequently, the effects of HV-107 and HV-118 were evaluated in cellsurvival and cell migration, known Rac-regulated cell functions relevantfor cancer progression. The effects of HV-107 and HV-118 on cancer cellsurvival were determined by measuring the levels of active caspase 3 inMDA-MB-231 and MDA-MB-435 metastatic breast cancer cells after treatmentwith HV-107 at 500 and 1000 nM, or HV-118 at 100 and 200 nM for 48-72 h.After 48 h, HV-107 caused a significant 1.4-1.5 fold increase in caspase3 activity in MDA-MB-231 (at 500 and 1000 nM) and MDA-MB-435 (at 1000nM) cells. Similar results were observed in cells treated with HV-118. A1.6-1.8 fold increase was demonstrated in caspase 3 activity inMDA-MB-231 cells after 48 h treatment with 100 and 200 nM HV-118;whereas, the same level of caspase 3 activity induction was observed inMDA-MB-435 cells at 72 h (FIG. 15 (A-D)). This data suggests that bothHV-107 and HV-118 inhibit cell survival by promoting apoptosis, themajor mechanism of programmed cell death. The effects of both compoundswere also examined in the activity of Akt, an important kinase thatpromotes survival signaling, is frequently hyperactivated in malignantcancers, and can be regulated by Rac. Because Akt is activated byphosphorylation events, the effects of HV-107 and HV-118 were analyzedin Akt phosphorylation by western blot (FIG. 16 (A-D)). Results showHV-107 at 250-1000 nM (for 24 h) significantly inhibits Aktphosphorylation by 50-60% in MDA-MB-231 cells; and causes a moderate 25%inhibition of Akt phosphorylation in MDA-MB-435 cells at 1000 nM. On theother hand, HV-118 significantly inhibits Akt phosphorylation by 40-60%in both MDA-MB-231 and MDA-MB-435 metastatic breast cancer cells treatedwith 100 and 200 nM for 24 h.

Finally, wound healing assays were performed to assess the effects ofHV-107 and HV-118 on metastatic breast cancer cell migration. Resultsshow cell migration was significantly inhibited by 30-50% at 2000 nMHV-107 and by approximately 60% and 80% at 100 and 200 nM HV-118,respectively in the metastatic breast cancer cell lines MDA-MB-231 andMDA-MB435 (FIG. 17 (A-B)). Taken together, these results confirm thatHV-107 and HV-118 significantly inhibit Rac regulated cell functions andAkt-mediated survival signaling in metastatic breast cancer cells.

An in vivo study was performed to assess HV-118 toxicity and test itseffects in mammary tumor growth and metastasis. Immunocompromised micewere inoculated with green fluorescent protein (GFP)-tagged MDA-MB-231cells. After tumors reached an average size of 80 mm³, mice (n=15/group)were administered with control or HV-118 at 1 mg/kg body weight (BW) byintraperitoneal (i.p.) injection every day from Monday to Friday forapproximately 2 months. The effects of HV-118 on tumor growth weremonitored weekly by in situ fluorescence image analysis (starting fromday 1 of treatment). Mammary tumor growth was quantified using Image Jsoftware, as the pixel intensity (integrated density) determined fromdigital tracing of the fluorescent tumor area at each imaging sessionand calculated as a function of the fluorescence intensity on day one oftreatment. At necropsy, blood was collected, and organs were dissectedand stored in liquid nitrogen. Metastatic foci were quantified forfluorescence intensity and area of fluorescence (lesion size) for eachorgan by Image J analysis of digital images from a fluorescence steromicroscope. Results show a 30% reduction in tumor growth by 1 mg/kg BWHV-118 and a significant reduction of >90% in metastasis to lung andliver (FIG. 18 (A-D)). Toxicity was assessed by: 1) performing ahematologic profile (complete blood count (CBC) analysis), 2) measuringserum levels of liver enzymes Alanine Aminotranferase (ALT), AspartateAminotransferse (AST), and Alkaline phosphatase (ALP), and 3) monitoringanimal weight throughout the study. Results showed no changes in any ofthe parameters measured by the hematologic profile, serum levels of ALT,AST, or ALP, or mice weights; thus, suggesting no toxicity by HV-118 atthe tested dose (FIG. 19 (A-C)).

PUBLICATIONS

The following publications are incorporated by reference to the extentthey relate materials and methods disclosed herein.

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The invention claimed is:
 1. A compound of formula,

wherein R¹ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; each R²is independently deuterium, halogen, —OH, —CN, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₆-C₁₀ aryl, —OR⁵, —C(O)OR⁵,—C(O)NR⁵R⁶, —NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆alkyl, —CO₂H, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl),—C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl); wherein eachhydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, and C₆-C₁₀ aryl is independently optionally substituted; Ais —O—, —S—, —N(R⁷)—, —N(R⁷)C(O)—, or —C(O)N(R⁷)—; Y is —N—; R³ is—NHR¹¹, —NR¹¹R¹², —C(O)C₁-C₆ alkyl, —C(O)R¹¹, or 3-to 7-memberedheterocycloalkyl; wherein each hydrogen atom in —C(O)C₁-C₆ alkyl or 3-to7-membered heterocycloalkyl is optionally substituted by deuterium,halogen, —OH, oxo, —CN, —OC₁-C₆ alkyl, —NH₂, —NH(C₁-C₆ alkyl),—NH(C₆-C₁₀ aryl), —NH(C₆-C₁₀ aryl)-N(C₁-C₆ alkyl)₂, —NHC(O)C₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)C₁-C₆ alkyl, —NHC(O)NH₂, —NHC(O)NHC₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)NH₂, —N(C₁-C₆ alkyl)C(O)NHC₁-C₆ alkyl, —NHC(O)N(C₁-C₆alkyl)₂, —N(C₁-C₆ alkyl)C(O)N(C₁-C₆ alkyl)₂, —NHC(O)OC₁-C₆ alkyl,—N(C₁-C₆ alkyl)C(O)OC₁-C₆ alkyl, —NHS(O)(C₁-C₆ alkyl), —NHS(O)₂(C₁-C₆alkyl), —N(C₁-C₆ alkyl)S(O)(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂(C₁-C₆alkyl), —NHS(O)NH₂, —NHS(O)₂NH₂, —N(C₁-C₆ alkyl)S(O)NH₂, —N(C₁-C₆alkyl)S(O)₂NH₂, —NHS(O)NH(C₁-C₆ alkyl), —NHS(O)₂NH(C₁-C₆ alkyl),—NHS(O)N(C₁-C₆ alkyl)₂, —NHS(O)₂N(C₁-C₆ alkyl)₂, —N(C₁-C₆alkyl)S(O)NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)S(O)₂NH(C₁-C₆ alkyl), —N(C₁-C₆alkyl)S(O)N(C₁-C₆ alkyl)₂, —N(C₁-C₆ alkyl)S(O)₂N(C₁-C₆ alkyl)₂, —CO₂H,—C(O)C₁-C₆ alkyl, —C(O)OC₁-C₆ alkyl, —C(O)NH₂, —C(O)NH(C₁-C₆ alkyl),—C(O)N(C₁-C₆ alkyl)₂, —SC₁-C₆ alkyl, —S(O)C₁-C₆ alkyl, —S(O)₂C₁-C₆alkyl, —S(O)NH(C₁-C₆ alkyl), —S(O)₂NH(C₁-C₆ alkyl), —S(O)N(C₁-C₆alkyl)₂, —S(O)₂N(C₁-C₆ alkyl)₂, —P(C₁-C₆ alkyl)₂, —P(O)(C₁-C₆ alkyl)₂,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3-to 7-memberedheterocycloalkyl), —CF₃, —CHF₂, or —CH₂F; wherein each of R¹¹ and R¹² isindependently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₆ cycloalkyl, C₁-C₆ heteroalkyl, 3- to 7-membered heterocycloalkyl,C₆-C₁₀ aryl, C₁-C₆alkyl-(3- to 7-membered heterocycloalkyl), heteroaryl,—NHC₁-C₆alkyl, —N(C₁-C₆ alkyl)₂, —S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or—CH₂F, wherein each hydrogen in C₁-C₆alkyl, C₁-C₆heteroalkyl, and C₆-C₁₀aryl is optionally substituted by —O—C₁-C₆alkyl, C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, 3- to 7-membered substitutedheterocycloalkyl, —CH₂CN, —N(C₁-C₆ alkyl)₂, optionally substituted aryl,—C(O)NH-optionally substituted aryl, hydroxy, or mono- or bicyclicheteroaryl; or R³ is —OR¹¹, wherein R¹¹ is C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₁-C₆ heteroalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆alkyl-(3-to 7-memberedheterocycloalkyl), heteroaryl, —NHC₁-C₆ alkyl, —N(C₁-C₆ alkyl)₂,—S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or —CH₂F, wherein each hydrogen in C₁-C₆alkyl, C₁-C₆ heteroalkyl, and C₆-C₁₀ aryl is optionally substituted byC₃-C₆ cycloalkyl, 3- to 7-membered heterocycloalkyl, 3- to 7-memberedsubstituted heterocycloalkyl, —CH₂CN, —N(C₁-C₆ alkyl)₂, optionallysubstituted aryl, —C(O)NH-optionally substituted aryl, hydroxy, or mono-or bicyclic heteroaryl R⁸ is H, deuterium, halo C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₁-C₆ heteroalkyl, 3- to7-membered heterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆alkyl-(3-to 7-memberedheterocycloalkyl), mono- or bicyclic heteroaryl, —OC₁-C₆ alkyl, —NHC₁-C₆alkyl, —N(C₁-C₆ alkyl)₂, —S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or —CH₂F,wherein each hydrogen in C₁-C₆alkyl, C₁-C₆ heteroalkyl, 3- to 7-memberedheterocycloalkyl, mono- or bicyclic heteroaryl, and C₆-C₁₀ aryl isoptionally substituted by —O—C₁-C₆alkyl, C₃-C₆ cycloalkyl, 3- to7-membered heterocycloalkyl, 3- to 7-membered substitutedheterocycloalkyl, —CH₂CN, —N(C₁-C₆ alkyl)₂, —OC₁-C₆ alkyl, optionallysubstituted aryl, —C(O)NH-optionally substituted aryl, hydroxy, or mono-or bicyclic heteroaryl; R¹⁰ is H, deuterium, halo, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, 3-to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, or mono- or bicyclic heteroaryl; whereineach hydrogen atom in C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, or mono- orbicyclic heteroaryl is independently optionally substituted; each R⁵,R⁶, and R⁷ is independently H, deuterium, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₁-C₆ heteroalkyl, 3- to 7-memberedheterocycloalkyl, C₆-C₁₀ aryl, C₁-C₆ alkyl-(3- to 7-memberedheterocycloalkyl), heteroaryl, —OC₁-C₆ alkyl, —NHC₁-C₆ alkyl, —N(C₁-C₆alkyl)₂, —S(O)₂C₁-C₆ alkyl, —CF₃, —CHF₂, or —CH₂F, wherein each hydrogenin C₁-C₆alkyl, C₁-C₆ heteroalkyl, and C₆-C₁₀ aryl is optionallysubstituted by —O—C₁-C₆alkyl, C₃-C₆ cycloalkyl, 3- to 7-memberedheterocycloalkyl, 3- to 7-membered substituted heterocycloalkyl, —CH₂CN,—N(C₁-C₆ alkyl)₂, —OC₁-C₆ alkyl, optionally substituted aryl,—C(O)NH-optionally substituted aryl, hydroxy, or mono- or bicyclicheteroaryl; and a is 0, 1, 2, or 3; or a salt thereof.
 2. A method oftreating breast cancer in a patient in need thereof the methodcomprising administering to the patient an effective amount of acompound according to claim
 1. 3. The method of claim 2, wherein thecompound reduces the expression of a Bcl-2 protein.
 4. The method ofclaim 2, wherein the breast cancer is mediated by a GTPase.
 5. Themethod of claim 4, wherein the GTPase is Rac 1 or Cdc42.
 6. The methodof claim 2, wherein the compound inhibits PAK1/2 activity.
 7. The methodof claim 2, wherein the compound inhibits STAT3 activity.
 8. The methodof claim 2, wherein the effective amount of the compound is in a rangeof about 0.01 mg/kg to about 100 mg/kg of body weight of the patient.